Mobile phase and method for separating ionizable organic compounds

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 Art

[0002] Chromatography techniques such as liquid chromatography and supercritical fluid chromatography have become indispensable as simple and precise methods for the separation and analysis of mixtures. In addition, the targets of separation and analysis by chromatography cover various substances including low-molecular-weight compounds, high-molecular-weight compounds, and biopolymers.

[0003] In the separation and analysis performed by these chromatography techniques, it is important to appropriately select both the stationary phase and the mobile phase according to the structure of the target analyte. Even if the selection of the stationary phase is appropriate, inappropriate selection of the mobile phase will cause problems such as: the target analyte is hardly retained by the stationary phase and elutes as a mixture, the analysis takes an extremely long time, or the target analyte remains retained on the stationary phase. Therefore, to achieve separation with the target accuracy within an acceptable time, it is necessary to select a mobile phase with appropriate design.

[0004] For example, when an ionizable organic compound such as an amine is used as the separation target, if the stationary phase and mobile phase are not appropriately selected, the separation target may not be sufficiently retained on the stationary phase, or even if it is retained on the stationary phase, the peak shape will be severely deformed, making it impossible to perform analysis, purification and other operations with the target accuracy. Examples of commonly used combinations of stationary phase and mobile phase for such separation targets include the combination of an ion exchanger and an electrolyte solution, the combination of a hydrophobic stationary phase and a weakly basic mobile phase (ion suppression mode), and the combination of a relatively hydrophobic stationary phase and a neutral to acidic mobile phase containing specific anions (ion pair mode).

[0005] As stationary phases used in ion-pair mode, stationary phases consisting of octadecylsilylated (ODS) silica gel or its homologues are widely known (Non-Patent Document 1). In addition, it is known that ion-pair mode is also useful for stationary phases comprising 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 is known, which contains an aqueous solution of one or more hydrophobic anion salts selected from the group consisting of chaotropic anion salts and hydrophobic organic acid salts. The ionized target to be separated is accompanied by the above-mentioned anion as a counterion nearby, and when it is retained by a hydrophobic stationary phase, the above-mentioned anion needs to undergo dehydration from a hydrated state. Therefore, the presence of hydrophobic anions with low hydration energy is considered advantageous for retaining the target to be separated by 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 [Overview of the project] [Problems that the invention aims to solve]

[0009] On the other hand, when using a mobile phase with a low water content, the anionic species mentioned above are not necessarily effective in retaining the target for separation on the stationary phase. However, depending on the target for separation or the purpose of separation, a mobile phase with a low water content may be preferable. Therefore, in order to broaden the range of 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 target for separation, especially ionizable organic compounds, on the stationary phase.

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

[0011] As a result of diligent research to solve the above problems, the present inventors have found that even with a mobile phase having a low water content, the above problems can be solved by incorporating a specific anion into the mobile phase. In other words, the gist of this disclosure is as follows.

[0012] [1] It contains one or more anions selected from chloride ions, bromide ions, and thiocyanate ions at a concentration of 1 mM to 300 mM. A mobile phase in which 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% by volume and 50% or less by volume, 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 countercation. [4] The mobile phase according to [3], wherein the counter cation is one or more selected from secondary ammonium ions, tertiary ammonium ions, and quaternary ammonium ions. [5] The mobile phase according to [3] or [4], further comprising an acid having a pKa of -2.0 or higher and 4.0 or lower in water at 25°C. [6] The mobile phase according to any one of [1] to [5], wherein the anion is a chloride ion. [7] A mobile phase used in liquid chromatography or supercritical fluid chromatography using a nonionic stationary phase (excluding size exclusion type stationary phase), as described in any of [1] to [6]. [8] The mobile phase according to [7], wherein the nonionic stationary phase is a stationary phase on which a ligand having a crown ether-like cyclic structure is supported on a support, or a stationary phase on which a polysaccharide derivative is supported on a support. [9] The process includes a separation step of separating ionizable organic compounds by liquid chromatography or supercritical fluid chromatography. The mobile phase used in the separation step is chloride ions, bromide ions, and thiocyanate. It contains one or more anions selected from ions at a concentration of 1 mM to 300 mM, and the solvent 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 50% by volume or less, an organic solvent, subcritical carbon dioxide, and supercritical carbon dioxide. A method for separating ionizable organic compounds, wherein the stationary phase used in the separation step is a nonionic stationary phase (excluding size exclusion type stationary phases).

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

[11] The separation method according to [9], wherein the anion forms a salt with a countercation.

[12] The separation method according to

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

[13] The separation method according to

[11] or

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

[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, there can be provided a mobile phase that has a low water content and is capable of increasing the retention of a desired separation target on a stationary phase. Furthermore, according to the present disclosure, there can be provided a method for favorably separating ionizable organic compounds such as amines by chromatography using the aforementioned mobile phase and a nonionic stationary phase. Brief Description of the Drawings

[0014] [Figure 1] It is a liquid chromatogram of dl-tryptophan in Example 1. [Figure 2] It is a liquid chromatogram of dl-tryptophan in Example 2. [Figure 3] It is a liquid chromatogram of dl-tryptophan in Example 3. [Figure 4] It is a liquid chromatogram of dl-tryptophan in Comparative Example 1. [Figure 5] It is a liquid chromatogram of dl-tryptophan in Comparative Example 2. [Figure 6] It is a liquid chromatogram of dl-tryptophan in Comparative Example 3. [Figure 7] It is a liquid chromatogram of dl-tyrosine in Example 4. [Figure 8] This is the liquid chromatogram of dl-tyrosine in Comparative Example 4. [Figure 9] This is the liquid chromatogram of dl-tryptophan in Example 5. [Figure 10] This is the liquid chromatogram of dl-tryptophan in Comparative Example 5. [Figure 11] This is a liquid chromatogram of the racemic mixture of 1-phenyl-1,2,3,4-tetrahydroisoquinoline in Example 6. [Modes for carrying out the invention]

[0015] The following describes specific embodiments of this disclosure, but each configuration and combination thereof in each embodiment is merely an example, and additions, omissions, substitutions, and other modifications are possible as appropriate, without departing from the spirit of this disclosure. This disclosure is not limited by the embodiments, but is limited only by the scope of the claims. Furthermore, each aspect disclosed herein can be combined with any other features disclosed herein. In this specification, when the lower and upper limits of a numerical range are described separately, the numerical range may be a combination of any lower and any upper limit. can.

[0016] 1. Mobile phase The first embodiment of this disclosure is 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, wherein 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 50% by volume or less, an organic solvent, subcritical carbon dioxide, and supercritical carbon dioxide. The mobile phase according to this embodiment may contain other components as long as they do not impede the separation performance. Furthermore, the mobile phase according to this embodiment is preferably a homogeneous system (single-phase system) at least under the conditions under which the analysis is performed. The mobile phase is a liquid that is flowed into the stationary phase together with the object to be separated in chromatography such as liquid chromatography and supercritical fluid chromatography, moves together with the object to be separated, and then elutes the separated product from the stationary phase.

[0017] To date, solutions containing chloride ions have been used as washing solutions to elute cations adsorbed on ion-exchange stationary phases, but their application to the mobile phase has not been considered. Furthermore, while there have been instances where trace amounts of hydrogen chloride were present in the mobile phase, this was not due to intentional addition of hydrogen chloride to improve separation performance, but rather to residual hydrochloric acid used for pH adjustment during buffer preparation. Therefore, the effects of incorporating chloride ions at a specific concentration into the mobile phase have not been investigated.

[0018] The inventors of this invention diligently investigated the effect of chloride ions on separation and found that a mobile phase containing chloride ions is extremely effective in increasing sample retention compared to a mobile phase that does not contain chloride ions. Chloride ions are not chaotropic ions, but rather cosmotropic ions, possessing properties opposite to those of chaotropic ions. Specifically, chloride ions have high hydration energy and exhibit strong interactions with water molecules. Therefore, from the perspective of forming ion pairs with ionized target molecules and adsorbing them onto the stationary phase, chloride ions are considered unfavorable. Nevertheless, it is surprising that including chloride ions in the mobile phase increases the retention of ionized target molecules (e.g., organic ammonium ions) on the stationary phase.

[0019] The inventors hypothesize the following reason why chloride ions increase retention of the target to be separated to the stationary phase, thereby improving separation performance. The solvent of the mobile phase according to this embodiment is one or more solvents selected from a water / organic solvent mixture mainly consisting of organic solvents, subcritical carbon dioxide, and supercritical carbon dioxide. In such solvents with a low water content (i.e., solvents with a water content of 50% by volume or less), chloride ions do not undergo sufficient hydration stabilization, and can easily form ion pairs with the ionized target to be separated. Furthermore, because the energy loss due to dehydration when incorporated into the hydrophobic stationary phase is small, they can be easily dehydrated. As a result, the target to be separated is well retained in the stationary phase. In addition, chloride ions are spherical, have low steric hindrance, and have high polarizability due to the outer shell electron orbitals characteristic of heavy atoms, and therefore have high affinity for organic compounds. For this reason, it is thought that by including chloride ions in the mobile phase, the target to be separated can be well retained in the stationary phase, and separation performance can be improved. Also, since bromide ions, which are halide ions, and thiocyanate ions, which are pseudohalide ions, have similar chemical properties to chloride ions, it is thought that they function similarly to chloride ions for the same 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 two or more may be used in any combination and ratio.

[0022] In the mobile phase, the (pseudo)halide ions may or may not form a salt with the counter cation. It is preferable that the (pseudo)halide ions form a salt with the counter cation in order to minimize damage such as corrosion to various components such as chromatographs and columns.

[0023] In this specification, "(pseudo)halide ions do not form a salt" 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, relative to 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; for example, it may be a method of mixing solutions obtained by dissolving each compound in a solvent with the other components, a method of mixing by bubbling hydrogen chloride gas or hydrogen bromide gas, or a method of directly mixing liquid thiocyanic acid with the other components. With regard to hydrogen chloride and hydrogen bromide, from the viewpoint of simplicity, it is preferable to employ a method of mixing a solution of hydrogen chloride or hydrogen bromide with the other components. For example, when preparing a mobile phase containing chloride anions at a concentration of 5 mM and using a water / acetonitrile = 5 / 95 (v / v) mixed solvent, it is preferable to first weigh out a predetermined amount of commercially available hydrochloric acid and, if necessary, mix it with acetonitrile to adjust the water / acetonitrile ratio to 5 / 95 (v / v), and then dilute it with the water / acetonitrile = 5 / 95 (v / v) mixed solvent so that the chloride ion concentration becomes 5 mM. However, when using a commercially available high-concentration aqueous solution (for example, 35% concentrated hydrochloric acid) as a (pseudo)halide ion source, since the water content in the high-concentration aqueous solution is low, the mobile phase may 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] Furthermore, thiocyanate ions are not commercially available because their free acid is unstable. Therefore, it is preferable to prepare the mobile phase containing thiocyanate ions by the method described later, namely, by mixing the thiocyanate salt with other components of the mobile phase. In other words, in the mobile phase according to this embodiment, it is preferable that the thiocyanate ions form a salt with a countercation.

[0026] Whether (pseudo)halide ions form salts in the mobile phase can be determined by quantitative analysis of (pseudo)halide ions, acid-base titration, and evaluation of the presence or absence of volatile residue. The quantitative analysis method for (pseudo)halide ions is not particularly limited, but can be performed by ion chromatography or inductively coupled plasma emission spectroscopy (IPC-AES). Acid-base titration is performed after diluting the mobile phase with water about 10 times to mitigate the influence 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 analysis, taking into account measurement accuracy, and furthermore, no crystalline residue containing (pseudo)halide ions is produced even after evaporation of the mobile phase to dryness (for example, to dryness at 30°C under a pressure of 10 hPa), then it can be determined that (pseudo)halide ions in the mobile phase do not form salts.

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

[0028] The countercation is not particularly limited as long as it is a cation other than a proton or hydronium ion, but typically it is an unsubstituted ammonium ion (NH4 +Examples include onium cations such as primary to quaternary ammonium ions; metal ions such as sodium ions and potassium ions; etc. The counteranion is preferably an onium cation. For example, when separating amines using a stationary phase on which a ligand having a crown ether-like cyclic structure is supported, onium cations are less likely to cause problems such as weakening the retention of ionized amines by interacting with the crown ether-like cyclic structure. Furthermore, the onium cation is preferably selected from secondary to quaternary ammonium ions, and is preferably selected from secondary and tertiary ammonium ions in order to be applicable to liquid chromatography-mass spectrometry.

[0029] Preferred examples of primary ammonium ions include monoalkylammonium ions such as methylammonium ions, ethylammonium ions, and butylammonium ions. Suitable examples of secondary ammonium ions include dialkylammonium ions such as dimethylammonium ions, diethylammonium ions, and dibutylammonium ions. Suitable examples of tertiary ammonium ions include trialkylammonium ions such as trimethylammonium ions, triethylammonium ions, and tributylammonium ions. Suitable examples of quaternary ammonium ions include tetraalkylammonium ions such as tetramethylammonium ions, tetraethylammonium ions, tetrabutylammonium ions, hexadecyltrimethylammonium ions, and octadecyltrimethylammonium ions. 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] Of the onium cations mentioned above, a salt of a (pseudo)halide ion and an unsubstituted ammonium ion (e.g., ammonium chloride) is preferable from the viewpoint of improving separation performance, but since its solubility in non-aqueous solvents is not high, it is preferable to include enough water in the solvent to dissolve the salt. Furthermore, when chromatography is performed using a stationary phase on which a ligand having a crown ether-like cyclic structure is supported, the salt may inhibit the separation target from being encapsulated by the ligand having a crown ether-like cyclic structure, so it is preferable to select another onium cation as the counteranion.

[0031] In embodiments where (pseudo)halide ions form salts with countercations in the mobile phase, it is desirable to control the pH of the mobile phase as needed.

[0032] For example, when separating amines by chromatography, if the amine exists in both free amine and ammonium ion states in the mobile phase, even a slight change in pH can drastically alter the ratio of free amine to ammonium ion, and consequently affect the behavior in chromatography. This leads to instability. Therefore, it becomes necessary to adjust the pH of the mobile phase to favor either the free amine or ammonium ion state.

[0033] Furthermore, when separating amines in ion-pair mode, the amine is converted to an ammonium ion, and its adsorption behavior is used for separation. If the amine is an alkylamine, the alkylamine is sufficiently protonated even if the mobile phase is neutral, so the above problem does not occur without adjusting the pH. However, if the amine is a weakly basic aromatic amine, it is desirable to add an acid to the mobile phase and adjust the pH to approximately 4.0 or below.

[0034] Furthermore, if the amine is an amino acid, the dissociation of the carboxyl group of the amino acid may weaken its retention to the stationary phase. Therefore, it is desirable to suppress the dissociation of the carboxyl group and protonate the amino group by lowering the pH to below 2.0.

[0035] Therefore, if (pseudo)halide ions form salts with countercations in the mobile phase, the pH is adjusted by adding acid as needed. This makes it possible to convert almost all amines into ammonium ions or to suppress the dissociation of 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 acid's pKa include, for example, -2.0 or higher and less than 4.0, -1.0 or higher and 3.8 or lower, and 0.5 or higher and 3.6 or lower. Acids stronger than hydrogen chloride convert chloride ions into hydrogen chloride, increasing the risk of metal corrosion, but this risk can be reduced if the pKa is -2.0 or higher. Furthermore, with acids that are too weak, it is difficult to protonate amino acids, but with a pKa of 4.0 or lower, it is possible to 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. Furthermore, when referring to the pKa of a polyhydric acid, it refers to pKa 1.

[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, formic acid and oxalic acid are particularly preferred due to their high acidity and low noise in detection by mass detectors. In addition to the above-mentioned acids, organic acids can also be used. When organic acids are added to the mobile phase, the signal-to-noise ratio at short wavelengths decreases slightly in detection by ultraviolet absorption, but this does not reduce the separation performance.

[0038] Furthermore, when adding acid to the mobile phase, if the acid is dissolved in a solvent of any choice before addition, the solvent composition of the mobile phase will change, disrupting the baseline of the chromatogram and leading to inaccurate analytical results. Therefore, it is preferable to use the same solvent for dissolving the acid as the solvent used in the mobile phase. The amount of acid added to the mobile phase should be appropriately selected according to the target to be separated and the solvent of the mobile phase, so that the pH of the mobile phase reaches the desired value. Typically, an amount of acid is added to the mobile phase such that the total concentration in the mobile phase is between 1 mM and 10 mM.

[0039] Whether or not (pseudo)halide ions are present in the mobile phase in the form of salts with countercations can be evaluated by the concentration of (pseudo)halide ions in the residue obtained by evaporating the mobile phase to dryness (for example, to dryness at 30°C under a pressure of 10 hPa). If the residue contains 0.1 mM or more of (pseudo)halide ions relative to the mobile phase volume, it can be presumed that (pseudo)halide ions are present in the mobile phase in the form of salts. Furthermore, if the countercation is a primary to quaternary ammonium ion, and a peak of an organic group bonded to the nitrogen atom of the ammonium ion is observed in the NMR spectrum of the residue, it can be presumed that (pseudo)halide ions are forming salts with primary to quaternary ammonium ions in the mobile phase. Also, if the countercation is a metal ion, and the presence of the metal ion is confirmed by ion chromatography, it can be presumed that (pseudo)halide ions are forming 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 also 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 strength of retention of the target to be separated on the stationary phase depends on the concentration of (pseudo)halide ions; retention increases with higher concentrations, but depending on conditions such as the solvent, the strength of retention reaches saturation at a certain concentration. Therefore, the concentration of (pseudo)halide ions does not need to be extremely high, and within the above range, the retention of the target to the stationary phase can be sufficiently increased. For example, in the examples described later, it is shown that even at low concentrations of chloride ions of 10 mM or less, the target to be separated is well retained on the stationary phase, resulting in good separation of the target. Furthermore, if (pseudo)halide ions, particularly chloride ions, are present in the mobile phase without forming salts, and the solvent of the mobile phase is a mixed solvent of water with a low water content and an organic solvent, or an organic solvent (with a water content of 0% by volume), there is a risk of corrosion of various components such as the chromatograph and columns. Therefore, if various components such as the chromatograph and columns are made of a material that is easily corroded, such as steel, it is desirable to keep the concentration of (pseudo)halide ions below 5 mM.

[0042] 1-2. Solvent The solvent of 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 50% or less by volume, an organic solvent (with a water content of 0% by volume), subcritical carbon dioxide, and supercritical carbon dioxide. In the above mixed solvent of water and an organic solvent and an organic solvent (with a water content of 0% by volume), the organic solvent may be a single type, or two or more types may be used in any combination and ratio.

[0043] The organic solvent included in the mixed solvent of water and an organic solvent is not particularly limited, but it is preferably an organic solvent that can dissolve the target to be separated. Suitable organic solvents include, for example, acetonitrile, methanol, ethanol, n-propanol, 2-propanol, tetrahydrofuran, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and dimethylacetamide (DMAc). In addition, the organic solvent may contain hydrocarbons such as hexane; ethers such as methyl tert-butyl ether (MTBE); halogenated hydrocarbons such as dichloromethane; etc., as long as it can dissolve the target to be separated and the various components such as the salts mentioned above. Of these, the organic solvent is preferably selected from acetonitrile, methanol, ethanol, n-propanol, 2-propanol, and tetrahydrofuran, more preferably from methanol and acetonitrile, and even more preferably from acetonitrile, in terms of high chemical stability, low viscosity, and the ability to detect amines by ultraviolet absorption. The organic solvent may also be a mixture of hydrocarbons having 5 to 8 carbon atoms and alcohols, which are commonly used in chromatography.

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

[0045] For the explanation of organic solvents with a water content of 0% by volume, refer to the explanation of organic solvents contained in a mixed solvent of water and an organic solvent.

[0046] When using a mobile phase in 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 salts of hydrogen chloride, hydrogen bromide, thiocyanic acid, or (pseudo)halide ions is not always good, it is preferable to use water or a water / methanol mixture as the solvent along with carbon dioxide so that the target (pseudo)halide ions dissolve in the mobile phase at the desired concentration.

[0047] 1-3. Items to be separated The mobile phase according to this embodiment is useful for ion-pair chromatography of ionizable organic compounds because it can ionize ionizable organic compounds by supplying (pseudo)halide ions to them and provide retention force to a nonionic stationary phase. By using the mobile phase according to this embodiment, it is possible to separate a mixture of multiple ionizable organic compounds into individual compounds, or to separate ionizable organic compounds from a mixture of ionizable organic compounds and other compounds.

[0048] Typical examples of ionizable organic compounds include amines. The amine is not particularly limited and may be a primary amine, a secondary amine, or a tertiary amine. Specific examples of amines include amino acids such as alanine, cysteine, glutamic acid, methionine, leucine, tyrosine, and tryptophan; amino acid derivatives such as esters of the above 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] By using the mobile phase according to this embodiment, the separation performance of chromatography can be improved, 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, mixtures of positional isomers, mixtures of geometric isomers, and mixtures of analogs. Furthermore, by performing chromatography using a chiral stationary phase, it is also possible to separate mixtures of enantiomers into individual enantiomers.

[0050] 1-4.Stationary phase The stationary phase to which the mobile phase according to this embodiment is applied is not particularly limited, but as described above, it is preferable that the stationary phase be nonionic in order to exhibit the characteristics of the mobile phase. Furthermore, since the size exclusion type stationary phase is used for separation based on differences in the molecular size of the molecules to be separated, and does not utilize chemical interactions between the stationary phase and the molecules to be separated, it is preferable that the mobile phase according to this embodiment be applied to a stationary phase other than the size exclusion type stationary phase.

[0051] In this specification, a nonionic stationary phase means a stationary phase that does not have ionic functional groups 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 ionic functional groups in the portion that comes into contact with the object to be separated. However, under the operating conditions, the stationary phase does not come into contact with the object to be separated. A stationary phase having a dipolar ionic functional group in the portion thereof, but lacking a positive or negative ionic functional group, is considered to be electrically neutral overall and is therefore included in the category of nonionic stationary phases.

[0052] The nonionic stationary phase is not particularly limited, and commercially available known stationary phases can be used. Known commercially available stationary phases include: stationary phases in which ligands having a crown ether-like cyclic structure are supported on a carrier; stationary phases in which polysaccharide derivatives such as cellulose derivatives and amylose derivatives are supported on a carrier; and stationary phases such as octadecylsilyl (ODS) silica gel, which are bonded to silica gel via alkylsilyl groups having 1 to 32 carbon atoms, or polar phases in which polar groups such as amide bonds are embedded in the alkyl chain of these alkylsilyl groups. Examples of stationary phases called embedded phases include: a stationary phase to which 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 having a cyano group (e.g., phenylpropyl group), a group having an amide bond (e.g., amidopropyl group), or a group having an alcoholic hydroxyl group is attached to silica gel; a stationary phase to which amino acids, cyclic oligosaccharides (e.g., cyclodextrin and cyclofructan), cyclic oligosaccharides modified to have hydroxyl groups, or macrocyclic amides (e.g., vancomycin) are supported on a carrier; a stationary phase having a dipolar ionic functional group; and so on.

[0053] Of the stationary phases described above, 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 in which a ligand having a crown ether-like cyclic structure is supported on a carrier. In this specification, a ligand means a compound that is supported on a carrier and exhibits physical affinity for the target to be separated, and, if necessary, asymmetric recognition ability. A 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 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) described later has n = 5 and has a crown ether-like cyclic structure of a size suitable for encapsulating a primary ammonium group, and is therefore suitably used for the separation of primary amines. The hydrogen atoms of the ethylene group in the repeating unit may be substituted with various functional groups, but it is preferable that they are not substituted.

[0056] In this embodiment, when separating enantiomers, a compound in which a crown ether-like cyclic structure is bonded to a homochiral structure is used as the ligand. Examples of such ligands include the ligand represented by formula (II) described in Japanese Patent Publication No. 2-69472 and International Publication No. 2012 / 050124, and the ligand represented by formula (III) described in Japanese Patent Publication No. 2014-169259. 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 the ligands 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] Ligands are used as a stationary phase in a state supported on a carrier. Known methods can be employed for support, and methods in which the ligand is supported on the carrier by chemical bonds such as covalent bonds are preferably employed. Specifically, one method involves introducing a reactive group into the ligand, the raw material for the ligand, or the intermediate for 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, or it may be a group introduced on the surface of the carrier by surface treating the carrier with a surface treatment agent, such as a silane coupling agent such as 3-aminopropyltriethoxysilane and 3-glycidyloxypropyltrimethoxysilane. In addition to forming a bond between the ligand and the carrier, it is also possible to form an insoluble layer on the surface of the carrier by forming so-called cross-linking bonds between atomic groups containing the ligand. Furthermore, the ligand may be supported on the carrier by other known support methods, such as physically adsorbing (coating) the ligand onto the carrier.

[0059] The support material is not particularly limited, as long as the ligand can be immobilized by chemical bonds such as covalent bonds. Such a support material may be inorganic or organic, but an inorganic support material is preferred. Examples of inorganic support materials include silica gel, alumina, magnesia, glass, kaolin, titanium dioxide, silicates, and hydroxyapatite. Examples of organic support materials include crosslinked polystyrene, crosslinked polyacrylamide, crosslinked polyacrylate, and polysaccharides. These organic support materials are preferably immobilized by crosslinking with a crosslinking agent.

[0060] The shape of the support is not particularly limited and can include, for example, particles and porous cylindrical bodies (monoliths) that are liquid-tightly housed in a column tube. The inner wall of a capillary can also be used as a support.

[0061] Furthermore, in this embodiment, the carrier is preferably silica gel. This is because silica gel has the aforementioned properties, namely excellent separation performance, and is also hard and durable. In addition to fully porous silica gel, 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 in which polysaccharide derivatives are supported on a carrier. As the stationary phase polysaccharide derivative supported on a carrier, known polysaccharide derivatives that can be used for the separation of optical isomers can be employed. Examples of known polysaccharide derivatives include cellulose, amylose, and β-1,4-xylan. Examples include polysaccharides selected from β-1,4-chitosan, chitin, β-1,4-mannan, inulin, or curdlan, in which the hydroxyl group is converted to benzoate, phenylcarbamate, 3,5-dimethylphenylcarbamate, 3-chloro-5-methylphenylcarbamate, 3,5-dichlorophenylcarbamate, 2,4-dichlorophenylcarbamate, 3,4-dichlorophenylcarbamate, 2,5-dichlorophenylcarbamate, 4-fluorophenylcarbamate, 4-chlorophenylcarbamate, 4-bromophenylcarbamate, or 4-iodophenylcarbamate. More specifically, examples include amylose (3-chloro-5-methylphenylcarbamate) described in Japanese Patent Publication No. 2018-054608; cellulose tris(3,5-dichlorophenylcarbamate) described in International Publication No. 2008 / 102920; cellulose trisbenzoate, cellulose tris(phenylcarbamate), and cellulose tris(3,5-dimethylphenylcarbamate) described in International Publication No. 2005 / 075974; and cellulose tris(4-chlorophenylcarbamate) described in International Publication No. 2002 / 030903.

[0063] Examples of carriers include those similar to stationary phase carriers, in which ligands having a crown ether-like cyclic structure are supported on the carrier.

[0064] Furthermore, known methods can be used to support the polysaccharide derivative on a carrier. Known methods include, but are not limited to, the methods described in Japanese Patent Publication No. 2018-054608, Japanese Patent Publication No. 2018-030965, International Publication No. 2014 / 087937, or Japanese Patent Publication No. Hei 7-138301.

[0065] 2. Method for separating ionizable organic compounds A second embodiment of this disclosure is a method for separating ionizable organic compounds, comprising a separation step of separating the 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 this disclosure. The stationary phase used in the separation step is a nonionic stationary phase (excluding size exclusion stationary phases). For descriptions of ionizable organic compounds and stationary phases, refer to the descriptions in "1-3. Objects to be Separated" and "1-4. Stationary Phase" above, respectively.

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

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

[0068] For the mass spectrometry step, known mass spectrometry methods used in LC-MS can be employed. For example, the ionization method in mass spectrometry can be appropriately selected from atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), electrospray method (ESI), fast atomic bombardment (FAB), and thermospray method (TSP) depending on the type of sample and the purpose of analysis. Furthermore, as for the mass spectrometer, quadrupole mass spectrometers (Q-MS), ion trap mass spectrometers (IT-MS), and time-of-flight mass spectrometers can be employed. It can be appropriately selected and used from analytical meters (TOF-MS), etc., depending on the required sensitivity and resolution.

[0069] 3. Use of (pseudo)halide ion-containing solutions as a mobile phase A third embodiment of this disclosure involves the use of a solution as a mobile phase, which contains 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 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 50% by volume or less, an organic solvent, subcritical carbon dioxide, and supercritical carbon dioxide. The solution in this embodiment is the mobile phase according to the first embodiment of this disclosure. That is, the anions, solvent, objects to be separated using the mobile phase, and stationary phase to which the mobile phase is applied in this embodiment are as described in "1-1. Anion Species," "1-2. Solvent," "1-3. Objects to be Separated," and "1-4. Stationary Phase," respectively. [Examples]

[0070] The present disclosure will be further described below with reference to examples, but the present disclosure is not limited to the following examples without departing from its essence.

[0071] [Example 1: Chiral isolation of dl-tryptophan] (Preparation of mobile phase) 5.00 mL of commercially available 1.00 N hydrochloric acid was weighed out using a volumetric pipette and added to a 1.00 L volumetric flask. After adding 74.4 g of acetonitrile to this hydrochloric acid, the solution was made up using a water / acetonitrile = 5 / 95 (v / v) mixed solvent to prepare mobile phase A containing hydrogen chloride at a concentration of 5 mM and a water / acetonitrile (5 / 95 (v / v)) mixed solvent.

[0072] (Separation of amines) A column packed with a chiral stationary phase in which a ligand having a crown ether-like cyclic structure represented by formula (IV) was supported on silica gel via chemical bonds (Daicel Corporation's "CROWNPAK CR-I(-)", inner diameter 3 mm, length 150 mm) was used as the column for amine separation. A high-performance liquid chromatography system (Shimadzu Corporation's "LC-20AD") was used as the liquid chromatography apparatus. For this separation, dl-tryptophan, the target of separation, was dissolved in a water / acetonitrile (1:1 (v / v)) mixed solvent containing approximately 5 mM hydrogen chloride to a concentration of approximately 0.1% w / v, and 2 μL of the resulting solution was injected into the column using an autosampler. Mobile phase 1 was delivered to the column, which was heated to 30°C, at a rate of 0.43 mL / min. 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 isolation of dl-tryptophan] (Preparation of mobile phase) Mobile phase B was prepared according to the method described in Example 1, containing trimethylammonium chloride at a concentration of 5 mM, oxalic acid at a concentration of 2.9 mM, and a water / acetonitrile (5 / 95 (v / v)) mixed solvent.

[0075] (Separation of amines) Chiral separation of dl-tryptophan was performed 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 Figure 2.

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

[0077] (Separation of amines) Chiral separation of dl-tryptophan was performed 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 Figure 3.

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

[0079] (Separation of amines) Chiral separation of dl-tryptophan was performed 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 Figure 4.

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

[0081] (Separation of amines) Chiral separation of dl-tryptophan was performed 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 Figure 5.

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

[0083] (Separation of amines) Chiral separation of dl-tryptophan was performed 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 Figure 6.

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

[0085] (Separation of amines) Chiral separation of dl-tyrosine was performed 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 7.

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

[0087] (Separation of amines) Chiral separation of dl-tyrosine was performed 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) Mobile phase E was prepared according to the method described in Example 1, containing triethylammonium chloride at a concentration of 5 mM, oxalic acid at a concentration of 10 mM, and a hexane / ethanol / water (100 / 100 / 4 (v / v / v)) mixed solvent.

[0089] (Separation of amines) Chiral separation of dl-tryptophan was performed 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 Figure 9.

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

[0091] (Separation of amines) Chiral separation of dl-tryptophan was performed in the same manner as in Example 1, except that mobile phase e was used instead of mobile phase A and the mobile phase flow rate was changed to 0.25 mL / min. The obtained chromatogram Grams are shown in Figure 10.

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

[0093] (Separation of amines) Chiral separation of dl-tryptophan was performed 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, inner diameter 4.6 mm, length 250 mm, manufactured by Daicel Corporation) in which cellulose tris(3,5-dimethylphenylcarbamate) was immobilized on a silica gel support via chemical bonding was used as the stationary phase, and the mobile phase flow rate was changed to 1.0 mL / min. The obtained chromatogram is shown in Figure 11.

[0094] Examples 1-5 and Comparative Examples 1-5 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 the chiral separation of primary amines, as it encapsulates primary ammonium ions in the crown ether-like cyclic structure through hydrogen bonding and recognizes the chirality of the sample through interaction with the chiral binaphthyl structure.

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

[0096] Conventionally, it has been known that when a mobile phase containing trifluoroacetic acid and an organic solvent such as acetonitrile or a hexane / ethanol mixed solvent as the main solvent is used, the ability to retain amines on the stationary phase is weak, and amines may not be separated well. In contrast, the mobile phases of Examples 1 to 5, despite being mainly organic solvents with a low water content, were found to retain amino acids more strongly on the stationary phase than the comparative examples above due to the inclusion of chloride ions. Furthermore, it was found that high retention performance was observed even at a low concentration of chloride ions in the mobile phase of 5 mM. On the other hand, while Comparative Example 5 showed separation performance comparable to Example 5, it was found that the concentration of trifluoroacetic acid required to obtain such separation performance (approximately 67 mM) was more than 13 times the concentration of chloride ions (5 mM) in Example 5.

[0097] Example 6 is an experimental example using a chiral stationary phase in which cellulose tris(3,5-dimethylphenylcarbamate) is immobilized on a silica gel support. When using such a stationary phase, it is known that problems arise in that ionic substances cannot be adequately retained or asymmetrically recognized when the mobile phase is mainly an organic solvent such as acetonitrile and has a low water content. In contrast, the results of Example 6 confirmed that even with a mobile phase mainly an organic solvent and with a low water content, good retention of amines and chiral separation can be achieved by adding triethylammonium chloride. [Industrial applicability]

[0098] By performing liquid chromatography using the mobile phase according to at least some embodiments of this disclosure, high amine separation performance can be achieved. Furthermore, by selecting highly volatile (pseudo)halide ions and acids, adverse effects on detection by mass spectrometers are suppressed, making it possible to apply this to LC-MS as well. Therefore, such mobile phases can be widely applied to analysis and purification using various liquid chromatography or supercritical fluid chromatography methods, and are expected to have applications in fields such as organic chemistry, medicine, and pharmaceuticals.

Claims

1. A mobile phase used in liquid chromatography using a nonionic stationary phase (excluding size exclusion type stationary phase), It contains chloride ions at a concentration of 1 mM to 300 mM. The chloride ions either do not form a salt, or they form a salt with tertiary ammonium ions. The solvent is a mixed solvent of water and an organic solvent having a water content of more than 0% by volume and 50% by volume or less, and one or more solvents selected from organic solvents. The mobile phase is a stationary phase in which a ligand having a crown ether-like cyclic structure is supported on a carrier, or a mobile phase in which a polysaccharide derivative is supported on a carrier.

2. The mobile phase according to claim 1, further comprising an acid having a pKa of -2.0 or more and 4.0 or less in water at 25°C.

3. The process includes a separation step of separating ionizable organic compounds by liquid chromatography. The mobile phase used in the separation step contains chloride ions at a concentration of 1 mM to 300 mM, and the chloride ions either do not form a salt or form a salt with tertiary ammonium ions. The solvent of the mobile phase is a mixed solvent of water and an organic solvent having a water content of more than 0% by volume and 50% by volume or less, and one or more solvents selected from organic solvents. The stationary phase used in the separation step is a nonionic stationary phase (excluding size exclusion type stationary phases). A method for separating ionizable organic compounds, wherein the nonionic stationary phase is a stationary phase on which a ligand having a crown ether-like cyclic structure is supported on a carrier, or a stationary phase on which a polysaccharide derivative is supported on a carrier.

4. The separation method according to claim 3, wherein the mobile phase further comprises an acid having a pKa of -2.0 or more and 4.0 or less in water at 25°C.

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