JP2023161874A - Mobile phase and method for separating ionizable organic compound - Google Patents

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

The present disclosure relates to mobile phases and methods for separating ionizable organic compounds.

Chromatography, such as liquid chromatography and supercritical fluid chromatography, has become indispensable as a simple and precise method for separating and analyzing mixtures. Furthermore, the targets of separation and analysis by chromatography include various substances such as low molecular weight compounds, high molecular weight compounds, and biopolymers.

In these chromatographic separation analyses, it is important to appropriately select both a stationary phase and a mobile phase depending on the structure to be separated. Even if the stationary phase is selected appropriately, if the mobile phase is inappropriately selected, for example, the target to be separated may be barely retained in the stationary phase and elute as a mixture, or the analysis may take an extremely long time. , the problem of remaining retained in the stationary phase occurs. Therefore, it is necessary to select a mobile phase of appropriate design in order to achieve the separation with the desired precision within an acceptable time.

For example, when an ionizable organic compound such as an amine is to be separated, if the stationary phase and mobile phase are not selected appropriately, the target to be separated may not be sufficiently retained in the stationary phase, or even if it is retained in the stationary phase. The peak shape may be severely deformed, making it impossible to perform analysis, purification, etc. with the desired accuracy. Combinations of stationary and mobile phases commonly used for such separations include a combination of an ion exchanger and an electrolyte solution, and 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).

As a stationary phase used in the ion pair mode, a stationary phase made of octadecylsilyl (ODS) silica gel or a homolog thereof is widely known (Non-Patent Document 1). In addition, it is known that the ion pair mode is useful for a stationary phase comprising a polysaccharide derivative and a stationary phase having a crown ether-like cyclic structure (Non-Patent Document 2, Patent Document 1).

The mobile phase to be combined with the stationary phase having a crown ether-like cyclic structure is a mobile phase mainly composed of water and one or more selected from the group consisting of salts of chaotropic anions and salts of hydrophobic organic acids. Mobile phases containing aqueous solutions of salts of hydrophobic anions are known. The ionized target to be separated has the above-mentioned anion as a counter ion in its vicinity, and when it is held on a hydrophobic stationary phase, the above-mentioned anion needs to be dehydrated from the hydrated state. It is believed that the presence of hydrophobic anions with low sum energy works advantageously to retain the separation target on the stationary phase (Patent Document 1).

International Publication No. 2020/251003

Analytical Chemistry, 2002, Vol.74, Issue19, p.4927-4932 Journal of Liquid Chromatography, 1993, Vol.16, Issue4, p.859-878

On the other hand, when using a mobile phase with a low water content, the above-mentioned anion species are not necessarily effective in effectively retaining the separation target in the stationary phase. However, depending on the separation target or separation purpose, a mobile phase with a low water content may be preferable. Therefore, in order to expand the options for analysis conditions in chromatography, it is necessary to develop a mobile phase with a low water content that can promote the retention of the desired separation target, especially ionizable organic compounds, on the stationary phase. is required.

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

As a result of intensive studies to solve the above problems, the present inventors have found that the above problems can be solved by incorporating a specific anion into the mobile phase even if the mobile phase has a low water content. . That is, the gist of the present disclosure is as follows.

[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,
The solvent is one or more solvents selected from a mixed solvent of water and an organic solvent, an organic solvent, subcritical carbon dioxide, and supercritical carbon dioxide, in which the water content is more than 0% by volume and not more than 50% by volume. Yes, mobile phase.
[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 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 more and 4.0 or less 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]
The mobile phase according to any one of [1] to [6], which is used in liquid chromatography or supercritical fluid chromatography using a nonionic stationary phase (excluding size exclusion type stationary phases).
[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]
comprising a separation step of separating ionizable organic compounds by liquid chromatography or supercritical fluid chromatography;
The mobile phase used in the separation step 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 has a water content of 0. One or more solvents selected from a mixed solvent of water and an organic solvent, an organic solvent, subcritical carbon dioxide, and supercritical carbon dioxide, which is more than 50 volume% by volume,
A method for separating ionizable organic compounds, wherein the stationary phase used in the separation step is a nonionic 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]
The separation method according to [9], wherein the anion forms a salt with a counter cation. [12]
The separation method according to [11], wherein the counter cation is one or more selected from secondary ammonium ions, tertiary ammonium ions, and quaternary ammonium ions.
[13]
The separation method according to [11] or [12], wherein the mobile phase further contains an acid whose pKa in water at 25° C. is −2.0 or more and 4.0 or less.
[14]
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. Separation method described in.

According to the present disclosure, it is possible to provide a mobile phase that has a low water content and can enhance the retention of a desired separation target on the stationary phase.
Further, according to the present disclosure, it is possible to provide a method for satisfactorily separating ionizable organic compounds such as amines by chromatography using the above mobile phase and a nonionic stationary phase.

1 is a liquid chromatogram of dl-tryptophan in Example 1. 2 is a liquid chromatogram of dl-tryptophan in Example 2. 3 is a liquid chromatogram of dl-tryptophan in Example 3. 1 is a liquid chromatogram of dl-tryptophan in Comparative Example 1. 2 is a liquid chromatogram of dl-tryptophan in Comparative Example 2. 3 is a liquid chromatogram of dl-tryptophan in Comparative Example 3. 3 is a liquid chromatogram of dl-tyrosine in Example 4. 3 is a liquid chromatogram of dl-tyrosine in Comparative Example 4. 3 is a liquid chromatogram of dl-tryptophan in Example 5. 3 is a liquid chromatogram of dl-tryptophan in Comparative Example 5. 1 is a liquid chromatogram of racemic 1-phenyl-1,2,3,4-tetrahydroisoquinoline in Example 6.

The present disclosure will be described below with reference to specific embodiments, but the configurations and combinations thereof in each embodiment are merely examples, and configurations may be modified as appropriate without departing from the gist of the present disclosure. addition, omission, substitution, and other changes are possible. This disclosure is not limited by the embodiments, but only by the scope of the claims.
Additionally, each aspect disclosed herein can be combined with any other feature disclosed herein.
In this specification, when a lower limit value and an upper limit value of a numerical range are described separately, the numerical range can be a combination of any lower limit value and any upper limit value among them.

1. Mobile Phase The first embodiment of the present 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 or more and 300 mM or less; The solvent contained in the phase is one selected from a mixed solvent of water and an organic solvent in which the water content is more than 0 volume % and 50 volume % or less, an organic solvent, subcritical carbon dioxide, and supercritical carbon dioxide. These are the above solvents. The mobile phase according to this embodiment may contain other components as long as the separation performance is not impaired. Further, the mobile phase according to the present embodiment is preferably a homogeneous system (single-phase system) at least under the conditions for performing analysis. In addition, in chromatography such as liquid chromatography and supercritical fluid chromatography, the mobile phase is a liquid that is poured into the stationary phase together with the separation target, moves together with the separation target, and then elutes the separated product from the stationary phase. .

Until now, a solution containing chloride ions has been used as a cleaning solution to elute cations adsorbed on an ion-exchange stationary phase, but the application of this solution to a mobile phase has not been considered. Additionally, there were cases in which trace amounts of hydrogen chloride were included in the mobile phase, but this was not due to hydrogen chloride being intentionally added to the mobile phase to improve separation performance, but rather due to the buffer solution being prepared. Hydrochloric acid used for pH adjustment remained in the mobile phase. Therefore, no study has been made as to what kind of effects can be obtained by containing chloride ions at a specific concentration in the mobile phase.

As a result of intensive studies on the influence of chloride ions on separation, the present inventors found that mobile phases containing chloride ions are extremely effective in increasing sample retention compared to mobile phases that do not contain chloride ions. I found it to be expensive.
Chloride ions are not classified as chaotropic ions, but as cosmotropic ions, which have the opposite properties to chaotropic ions. That is, chloride ions have large hydration energy and exhibit strong interactions with water molecules. Therefore, chloride ions are considered to be disadvantageous ions from the viewpoint of forming ion pairs with the ionized separation target and being adsorbed on the stationary phase. Nevertheless, it is surprising that the inclusion of chloride ions in the mobile phase has the effect of increasing the retention of ionized separation targets (for example, organic ammonium ions) in the stationary phase.

The present inventors speculate that the reason why chloride ions increase the retention of the separation target in the stationary phase and improve the separation performance is as follows.
The mobile phase solvent according to the present embodiment is one or more solvents selected from a water/organic solvent mixture containing an organic solvent as a main ingredient, subcritical carbon dioxide, and supercritical carbon dioxide. In a solvent with such a low water content (i.e., a solvent with a water content of 50% or less by volume), chloride ions do not undergo sufficient hydration stabilization and are easily separated from the ionized target. It can form ion pairs, and it can be easily dehydrated because there is little energy loss due to dehydration when it is incorporated into a hydrophobic stationary phase. As a result, the separation target is well retained on the stationary phase. In addition, chloride ions are spherical, have little steric hindrance, and have high polarizability due to the outer shell electron orbits unique to heavy atoms, so they have high affinity with organic compounds. Therefore, it is considered that by containing chloride ions in the mobile phase, the separation target can be retained well in the stationary phase and the separation performance can be improved. Furthermore, bromide ion, which is a halide ion, and thiocyanate ion, which is pseudohalide ion, have similar chemical properties to chloride ion, and therefore are thought to function similarly to chloride ion for the same reason.

Hereinafter, the mobile phase according to this embodiment will be explained in more detail.

1-1. Anion Species The mobile phase according to the present embodiment contains an anion selected from chloride ions, bromide ions, and thiocyanate ions (hereinafter sometimes referred to as "(pseudo)halide ions"). Among these, the (pseudo)halide ion is preferably a chloride ion. One type of (pseudo) halide ion may be used alone, or two or more types may be used in combination in any combination and ratio.

In the mobile phase, the (pseudo)halide ion may or may not form a salt with a counter cation. It is preferable that the (pseudo) halide ion forms a salt with a counter cation, since it is less likely to cause damage such as corrosion to various members such as a chromatograph and a column.

In this specification, (pseudo)halide ions do not form a salt when the content of cations other than protons and hydronium ions in the mobile phase is 10 mol% or less of (pseudo)halide ions, This means that it is preferably 5 mol% or less, more preferably 3 mol% or less, and even more preferably 1 mol% or less.

The mobile phase in which (pseudo)halide ions are present in the system without forming a salt is a mixture of a free acid selected from hydrogen chloride, hydrogen bromide, and thiocyanic acid with other components of the mobile phase. It can be prepared by The mixing method is not particularly limited, and for example, it may be a method of mixing a solution obtained by dissolving each compound in a solvent with other components, or a method of mixing by bubbling hydrogen chloride gas or hydrogen bromide gas. Alternatively, a method may be used in which liquid thiocyanic acid is mixed as it is with other components. Regarding hydrogen chloride and hydrogen bromide, from the viewpoint of simplicity, it is preferable to adopt a method of mixing a solution of hydrogen chloride or hydrogen bromide with other components. For example, when preparing a mobile phase containing chloride anion at a concentration of 5 mM and using a mixed solvent of water/acetonitrile = 5/95 (v/v), first, weigh out a predetermined amount of commercially available hydrochloric acid, If necessary, adjust the water/acetonitrile ratio to 5/95 (v/v) by mixing with acetonitrile, and then mix water/acetonitrile = 5/95 (v/v) so that the chloride ion concentration is 5 mM. v) A method of diluting with a mixed solvent is preferred. However, when using a commercially available high-concentration aqueous solution (e.g., 35% concentrated hydrochloric acid) as a (pseudo)halide ion source, the water/organic The mobile phase may be prepared by directly diluting the highly concentrated aqueous solution with the mobile phase solvent without adjusting the solvent ratio.

Note that thiocyanate ion is unstable as a free acid and is not commercially available. Therefore, it is desirable that a mobile phase containing thiocyanate ions be prepared by the method described below, that is, a method of mixing thiocyanate and other components of the mobile phase. In other words, in the mobile phase according to this embodiment, it is desirable that the thiocyanate ion forms a salt with a counter cation.

Whether (pseudo)halide ions do not form salts 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 method for quantifying (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 about 10 times as much water in order to alleviate the influence of the solvent composition. The concentration of (pseudo)halide ions determined by acid-base titration was equivalent to the concentration of (pseudo)halide ions determined by the above-mentioned quantification, taking into account measurement accuracy, and the mobile phase was evaporated to dryness ( For example, if a crystalline residue containing (pseudo)halide ions does not occur even after drying at 30°C under a pressure of 10 hPa, then the (pseudo)halide ions in the mobile phase have not formed a salt. It can be determined that

The mobile phase that exists in the system with (pseudo) halide ions forming a salt with a counter cation is a mixture of the salt of (pseudo) halide ions and counter cation with other components of the mobile phase. It can be prepared by The mixing method is not particularly limited, and for example, it may be a method in which each compound is mixed as it is with other components, or a method in which a solution obtained by dissolving each compound in a solvent is mixed with other components. Good too.

The counter cation is not particularly limited as long as it is a cation other than protons and hydronium ions, but typically onium cations such as unsubstituted ammonium ions (NH ₄ ⁺ ) and primary to quaternary ammonium ions; sodium ions and potassium ions. metal ions such as; and the like. Preferably, the counter anion is an onium cation. For example, when separating amines using a stationary phase in which a ligand with a crown ether-like cyclic structure is supported on a carrier, onium cations interact with the crown ether-like cyclic structure and weaken the retention of ionized amines. This is because defects are less likely to occur. 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 terms of its applicability to liquid chromatography-mass spectrometry. Preferably.

Preferred examples of the primary ammonium ion 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.
Preferred examples of the tertiary ammonium ion include trialkylammonium ions such as trimethylammonium ion, triethylammonium ion, and tributylammonium ion.
Preferred 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, and It is more preferably an alkyl group having 1 to 10 carbon atoms, and even more preferably an alkyl group having 1 to 4 carbon atoms.

Among the onium cations mentioned above, salts of (pseudo)halide ions and unsubstituted ammonium ions (e.g. ammonium chloride) are suitable from the viewpoint of improving separation performance, but because they do not have high solubility in non-aqueous solvents, It is preferable that the solvent contains enough water to dissolve the salt. In addition, when performing chromatography using a stationary phase in which a ligand having a crown ether-like cyclic structure is supported on a carrier, the salt prevents the target to be separated from being included in the ligand having a crown ether-like cyclic structure. Therefore, it is preferable to select another onium cation as the counter anion.

In embodiments where the (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.

For example, when separating amines by chromatography, if the amine exists in both the free amine and ammonium ion states in the mobile phase, a slight change in pH will greatly change the abundance ratio of free amines and ammonium ions, resulting in a chromatographic separation. Behavior in graphics becomes unstable. Therefore, it is necessary to adjust the pH of the mobile phase in order to bias it toward either the free amine or ammonium ion state.

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 amine is an alkylamine, the alkylamine is sufficiently protonated even if the mobile phase is neutral, so the above problem does not occur even if the pH is not adjusted. However, when the amine is a weakly basic aromatic amine, it is desirable to add an acid to the mobile phase to adjust the pH to approximately 4.0 or less.

Furthermore, when the amine is an amino acid, the retention of the separation target on the stationary phase may be weakened due to dissociation of the carboxy group of the amino acid. Therefore, it is desirable to suppress dissociation of carboxy groups and protonate amino groups by controlling the pH to less than 2.0.

Therefore, when the (pseudo) halide ion forms a salt with a counter cation in the mobile phase, the pH is adjusted by adding an acid as necessary. This makes it possible to convert almost all amines into ammonium ions and to suppress the dissociation of carboxy groups of amino acids.

The pKa of the acid used for adjusting the pH of the mobile phase in water at 25°C is usually -2.0 or more, preferably -1.0 or more, more preferably 0.5 or more, and usually 4.0 or less. , preferably less than 4.0, more preferably 3.8 or less, still more preferably 3.6 or less. Preferred ranges for the pKa of the acid include, for example, -2.0 or more and less than 4.0, -1.0 or more and 3.8 or less, and 0.5 or more and 3.6 or less. Acids stronger than hydrogen chloride convert chlorine ions into hydrogen chloride, increasing the risk of metal corrosion; however, if the pKa is -2.0 or higher, this risk can be reduced. Furthermore, it is difficult to protonate amino acids with too weak acids, but if the pKa is 4.0 or less, it is possible to protonate almost all amino acids.
In this specification, the "pKa" of an acid means the pKa in water at 25° C. unless otherwise specified. Furthermore, when referring to the pKa of a polyvalent acid, the pKa shall mean pKa1.

Preferred examples of such acids include phosphoric acid (pKa: 2.15), oxalic acid (pKa: 1.04), and formic acid (pKa: 3.55). Among these, it is particularly preferable that the acid is selected from formic acid and oxalic acid in terms of high acidity and low noise in detection with a mass detector. Furthermore, in addition to the above-mentioned acids, it is also possible to use organic acids. When an organic acid is added to the mobile phase, the S/N ratio at short wavelengths is slightly lowered in ultraviolet absorption detection, but this does not lower the separation performance.

Note that when adding acid to the mobile phase, if the acid is dissolved in any solvent and added, if the solvent composition of the mobile phase changes, the baseline of the chromatogram will be disturbed and the analysis results will be inaccurate. The solvent for dissolving the acid is preferably the same as the solvent for the mobile phase.
The amount of acid added to the mobile phase may be appropriately selected depending on the object to be separated and the solvent of the mobile phase so that the pH of the mobile phase becomes a desired value. Usually, an amount of acid is added to the mobile phase such that the total concentration in the mobile phase is 1 mM or more and 10 mM or less.

Whether or not the mobile phase contains (pseudo)halide ions in the form of a salt with a counter cation can be determined by evaporating the mobile phase to dryness (for example, drying it at 30°C under a pressure of 10 hPa). The concentration of (pseudo)halide ions in the resulting residue can be evaluated. If the residue contains 0.1 mM or more (pseudo) halide ions based on the volume of the mobile phase, it can be assumed that the (pseudo) halide ions are contained in the mobile phase in the form of a salt. . In addition, when the counter cation is a primary to quaternary ammonium ion, if 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 is possible to detect a (pseudo) halide in the mobile phase. It can be assumed that the ions form a salt of primary to quaternary ammonium ions. In addition, when the counter cation is a metal ion, if the presence of the metal ion is confirmed by ion chromatography, it can be assumed that the (pseudo)halide ion is forming a salt with the metal ion in the mobile phase. .

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

The strength of retention on the stationary phase of the separation target depends on the concentration of (pseudo)halide ions, and the higher the concentration, the stronger the retention, but depending on the conditions such as the solvent, the strength of retention may vary at a constant concentration. Reach saturation. Therefore, the concentration of (pseudo)halide ions does not need to be extremely high, and as long as it is within the above range, retention in the stationary phase to be separated can be sufficiently increased. For example, in the Examples described below, it has been shown that even if the chloride ion concentration is as low as 10 mM or less, the target to be separated can be well separated as a result of the target being well retained in the stationary phase.
Note that (pseudo)halide ions, especially chloride ions, are contained in the mobile phase without forming a salt, and the mobile phase solvent is a mixed solvent of water and an organic solvent with a low water content or an organic solvent. If it is a solvent (water content: 0% by volume), there is a risk of corroding various members such as a chromatograph and a column. Therefore, when various members such as a chromatograph and a column are made of a material that is easily corroded, such as steel, it is desirable that the concentration of (pseudo) halide ions is 5 mM or less.

1-2. Solvent The solvent of the mobile phase according to this embodiment is a mixed solvent of water and an organic solvent with a water content of more than 0 volume % and 50 volume % or less, an organic solvent (water content 0 volume %), a subcritical One or more solvents selected from carbon dioxide and supercritical carbon dioxide. In the mixed solvent of water and organic solvent and the organic solvent (water content 0% by volume), the organic solvent may be used alone or in combination of two or more in any combination and ratio. good.

The organic solvent contained in the mixed solvent of water and organic solvent is not particularly limited, but is preferably an organic solvent that can dissolve the object to be separated. Suitable organic solvents include, for example, acetonitrile, methanol, ethanol, n-propanol, 2-propanol, tetrahydrofuran, dimethylsulfoxide (DMSO), dimethylformamide (DMF), and dimethylacetamide (DMAc). In addition, as long as the organic solvent can dissolve various components such as the target of separation and the above-mentioned salts, the organic solvent may contain hydrocarbons such as hexane; ethers such as methyl tert-butyl ether (MTBE); halogenated hydrocarbons such as dichloromethane; Good too. Among these, organic solvents are acetonitrile, methanol, ethanol, n-propanol, 2-propanol, and tetrahydrofuran because they have high chemical stability, low viscosity, and enable detection of amines by ultraviolet absorption. It is preferably selected from methanol and acetonitrile, and even more preferably acetonitrile. Further, 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.

The content of water in the mixed solvent of water and organic solvent is usually more than 0 vol%, preferably 0.5 vol% or more, more preferably 1.0 vol% or more, and usually 50 vol% or less, The content is preferably 30% by volume or less, more preferably 20% by volume or less, even more preferably 10% by volume or less. The preferable range of the water content in the mixed solvent of water and organic solvent is more than 0 vol% and 30 vol% or less, 0.5 vol% or more and 20 vol% or less, and 1.0 vol% or more and 10 vol% or less. can be mentioned.

For the description of the organic solvent with a water content of 0% by volume, the description of the organic solvent contained in the mixed solvent of water and organic solvent will be referred to.

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, the compatibility of hydrogen chloride, hydrogen bromide, thiocyanate, or (pseudo) halide ion salts with carbon dioxide is not necessarily good, so the desired (pseudo) halide ions are present in the mobile phase at the desired concentration. It is preferred to use water or a water/methanol mixture as a solvent, together with carbon dioxide, so that the solvent is dissolved in water.

1-3. Target of separation The mobile phase according to this embodiment ionizes the ionizable organic compound by supplying (pseudo) halide ions to the ionizable organic compound, and increases the retention force against the nonionic stationary phase. This makes it useful for ion pair chromatography of ionizable organic compounds. By using the mobile phase according to this embodiment, it is possible to separate a mixture of a plurality of ionizable organic compounds into each compound, or to separate an ionizable organic compound from a mixture of an ionizable organic compound and another compound. You can

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 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; cyclic amines such as 1-phenyl-1,2,3,4-tetrahydroisoquinoline; and the like.

By using the mobile phase according to the present embodiment, the separation performance of chromatography can be improved, so that it is also possible to separate a mixture of amines that are difficult to separate into individual amines because of their mutually similar structures. Examples of the mixture of amines having similar structures include a mixture of chain isomers, a mixture of positional isomers, a mixture of geometric isomers, a mixture of analogs, and the like. Furthermore, a mixture of enantiomers can be separated into each enantiomer by performing chromatography using a chiral stationary phase.

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, in order to exhibit the characteristics of the mobile phase, it is preferably a nonionic stationary phase. In addition, the size exclusion type stationary phase is used for separation based on the difference in the molecular size of the separation target, and does not utilize the chemical interaction between the stationary phase and the separation target. The mobile phase according to the configuration is preferably applied to a stationary phase other than a size exclusion type stationary phase.

In addition, in this specification, a nonionic stationary phase means a stationary phase that does not have an ionic functional group in the part that is in contact with the separation target. On the other hand, an ionic stationary phase means a stationary phase that has an ionic functional group in a portion that comes into contact with the separation target. However, under the usage conditions, a stationary phase that has a zwitterionic functional group in the part that comes into contact with the separation target and does not have a positive or negative ionic functional group is charge-neutral as a whole, so it is nonionic. shall be included in the stationary phase.

The nonionic stationary phase is not particularly limited, and commercially known stationary phases can be used.
Commercially known stationary phases include a stationary phase in which a ligand having a crown ether-like cyclic structure is supported on a carrier; a stationary phase in which a polysaccharide derivative such as a cellulose derivative and an amylose derivative is supported on a carrier; and octadecylsilyl (ODS). ) A stationary phase bonded to silica gel through an alkylsilyl group having 1 to 32 carbon atoms, such as silica gel, or a stationary phase called Polar Embedded, in which a polar group such as an amide bond is embedded in the alkyl chain of this alkylsilyl group. Stationary phase; silica gel containing a group containing a benzene ring (e.g., phenylethyl group and phenylhexyl group), a halogen-containing group (e.g., pentafluorophenylpropyl, pentachlorophenylpropyl, pentabromophenylpropyl, etc.), and a cyano group A stationary phase to which a group (e.g., phenylpropyl group, etc.), a group having an amide bond (e.g., amidopropyl group, etc.), or a group having an alcoholic hydroxyl group is bonded; Examples include a stationary phase in which a carrier supports a cyclic oligosaccharide (such as fructan), a cyclic oligosaccharide with its hydroxyl group modified, or a macrocyclic amide (for example, vancomycin, etc.); a stationary phase having a zwitterionic functional group; and the like.

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 explained in more detail below.

1-4-1. A stationary phase in which a ligand having a crown ether-like cyclic structure is supported on a carrier. In this specification, the term "ligand" refers to a ligand that is supported on a carrier and has a physical affinity for the separation target and, if necessary, an asymmetric It means a compound that exhibits recognition ability. A ligand having a crown ether-like cyclic structure has a macrocyclic polyether structure in which the crown ether skeleton represented by formula (I) is chemically bonded to an aliphatic, alicyclic, or aromatic hydrocarbon. This is the compound formed.

*-O(CH ₂ CH ₂ O) _n -* (I)
In the formula, n can be appropriately selected from an integer of 4 to 6 depending on the hydrocarbon to which the amino group of the amine and the crown ether skeleton are bonded. For example, the ligand represented by formula (II) or (III) described below has a crown ether-like cyclic structure in which n is 5 and has a size suitable for encompassing a primary ammonium group, and thus a primary amine It is suitably used for the separation of The hydrogen atom of the ethylene group in the repeating unit may be substituted with various functional groups, but is preferably not substituted.

When separating enantiomers in this embodiment, a compound in which a crown ether-like cyclic structure is bonded to a homochiral structure is used as a ligand. Examples of such a ligand include the ligand represented by the formula (II) described in JP-A-2-69472 and WO 2012/050124, and the formula (II) described in JP-A-2014-169259. Examples include the ligand represented by III). Further, the phenyl group at the 3-position and 3'-position of the 1,1'-binaphthyl structure in formula (II) is a halogen atom such as a bromine atom; an alkyl group such as a methyl group; a substituted aromatic group; a heterocyclic group; (Peng Wu, et.al., Chin. J. Chem., 2017, 35, 1037-1042) can also be employed, but the ligand is not limited to these. Furthermore, ligands that are effective for separating enantiomers are often also effective for separating molecules that are similar to each other.

The ligand is used as a stationary phase while supported on a carrier. As a method for supporting, a known method can be employed, and for example, a method in which the ligand is supported on a carrier by a chemical bond such as a covalent bond can be suitably employed. Specifically, a method may be mentioned in which a reactive group is introduced into a ligand, a raw material of the ligand, or an intermediate of the ligand, and this substituent is reacted with a reactive group present on the surface of the carrier. Note that the reactive group present on the carrier surface may be a group present on the surface of an untreated carrier, and the carrier may be treated with a surface treatment agent such as 3-aminopropyltriethoxysilane and 3-glycidyloxypropyl. It may also be a group introduced onto the surface of the carrier by surface treatment with a silane coupling agent such as trimethoxysilane. Furthermore, in addition to the method of forming a bond between a ligand and a carrier, it is also possible to form an insolubilized layer on the carrier surface by forming a so-called cross-linking bond between atomic groups containing the ligand. Furthermore, the ligand may be supported on the carrier by other known supporting methods, such as a method of physically adsorbing (coating) the ligand onto the carrier.

The carrier is not particularly limited as long as the ligand can be immobilized 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 preferable. Examples of the inorganic carrier include silica gel, alumina, magnesia, glass, kaolin, titanium oxide, silicate, and hydroxyapatite. Examples of the organic carrier include crosslinked polystyrene, crosslinked polyacrylamide, crosslinked polyacrylate, and polysaccharide. These organic carriers are preferably insolubilized by being crosslinked with a crosslinking agent.

The shape of the carrier is not particularly limited, and examples thereof include particles, a porous cylindrical body (monolith) accommodated in a column tube in a liquid-tight manner, and the like. Moreover, the inner wall of a capillary can also be used as a carrier.

Furthermore, in this embodiment, the carrier is preferably silica gel. This is because silica gel has the above-mentioned characteristics, that is, 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, and silica gel whose surface has been chemically modified may also be used.

1-4-1. Stationary phase in which a polysaccharide derivative is supported on a carrier As the polysaccharide derivative in the stationary phase in which a polysaccharide derivative is supported on a carrier, known polysaccharide derivatives that can be used for separation of optical isomers can be employed.
Known polysaccharide derivatives include, for example, polysaccharides selected from cellulose, amylose, β-1,4-xylan, β-1,4-chitosan, chitin, β-1,4-mannan, inulin, or curdlan. The hydroxyl group is replaced by 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 Examples include those converted to -dichlorophenyl carbamate, 4-fluorophenyl carbamate, 4-chlorophenyl carbamate, 4-bromophenyl carbamate, or 4-iodophenyl carbamate. More specifically, amylose (3-chloro-5-methylphenyl carbamate) described in JP 2018-054608; cellulose tris (3,5-dichlorophenyl carbamate) described in International Publication No. 2008/102920; Cellulose tris benzoate, cellulose tris (phenyl carbamate), and cellulose tris (3,5-dimethylphenyl carbamate) described in WO 2005/075974; cellulose tris (4-chlorophenyl carbamate) described in WO 2002/030903 ; etc. are exemplified.

Examples of the carrier include carriers similar to those of the stationary phase in which a ligand having a crown ether-like cyclic structure is supported on the carrier.

Further, as a method for supporting the polysaccharide derivative on a carrier, a known method can be employed. Known methods include, for example, the methods described in JP 2018-054608, JP 2018-030965, WO 2014/087937, or JP 7-138301; It is not limited.

2. Method for separating ionizable organic compounds A second embodiment of the present disclosure is a method for separating ionizable organic compounds, which includes a separation step of separating ionizable organic compounds by liquid chromatography or supercritical fluid chromatography. be. The mobile phase used in the separation step is the mobile phase according to the first embodiment of the present disclosure. Furthermore, the stationary phase used in the separation step is a nonionic stationary phase (excluding size exclusion type stationary phases). Regarding the explanation of the ionizable organic compound and the stationary phase, the explanations in "1-3. Target of separation" and "1-4. Stationary phase" above are referred to, respectively.

In addition to the separation step, the separation method according to the present embodiment may include other steps such as a step of qualitatively determining the separated sample and a step of quantifying the separated sample.
Liquid chromatography and supercritical fluid chromatography can be performed using commercially available liquid chromatographs and supercritical fluid chromatographs, respectively. Various conditions such as column equilibration conditions and flow rate can be appropriately selected depending on the column size, sample volume, type of mobile phase, etc.

Furthermore, the separation method according to the present embodiment may be applied to liquid chromatography-mass spectrometry (LC-MS) to perform various analyzes such as quantitative analysis and qualitative analysis of the separated sample. The analysis method using LC-MS includes a separation step in which an ionizable organic compound is separated by the separation method according to the present embodiment, and a mass spectrometry step in which the sample separated in the separation step is analyzed by mass spectrometry.

As the mass spectrometry in the mass spectrometry step, a known mass spectrometry method used in LC-MS can be employed. For example, ionization in mass spectrometry includes atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), electrospray method (ESI), fast atom bombardment method (FAB), thermospray method (TSP), etc. It can be selected as appropriate depending on the type of sample, purpose of analysis, etc. It is also required as a mass spectrometer, such as quadrupole mass spectrometers (Q-MS), ion trap mass spectrometers (IT-MS), and time-of-flight mass spectrometers (TOF-MS). They can be appropriately selected and used depending on the sensitivity and resolution.

3. Use of a (pseudo)halide ion-containing solution as a mobile phase The third embodiment of the present disclosure provides a solution 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. The solvent is selected from a mixed solvent of water and an organic solvent, organic solvent, subcritical carbon dioxide, and supercritical carbon dioxide, in which the water content is more than 0 volume % and 50 volume % or less. The use of a solution of more than one solvent as a mobile phase. The solution in this embodiment is the mobile phase according to the first embodiment of the present disclosure. That is, in this embodiment, the anion, the solvent, the target to be separated using the mobile phase, and the stationary phase to which the mobile phase is applied are the above-mentioned "1-1. anion species", "1-2. solvent", and "1-2. solvent", respectively. 1-3. Target of separation” and “1-4. Stationary phase”.

Hereinafter, the present disclosure will be explained in more detail with reference to examples, but the present disclosure is not limited to the following examples unless it departs from the gist thereof.

[Example 1: Chiral separation of dl-tryptophan]
(Preparation of mobile phase)
5.00 mL of commercially available 1.00N hydrochloric acid was weighed out using a whole pipette and added to a 1.00 L volumetric flask. After adding 74.4 g of acetonitrile to this hydrochloric acid, it was diluted with a mixed solvent of water/acetonitrile = 5/95 (v/v) to contain hydrogen chloride at a concentration of 5 mM, and water/acetonitrile was used as the solvent. Mobile phase A containing a (5/95 (v/v)) mixed solvent was prepared.

(Separation of amine)
A column packed with a chiral stationary phase in which a ligand having a crown ether-like cyclic structure represented by formula (IV) is supported on silica gel through a chemical bond ("CROWNPAK CR-I (-)" manufactured by Daicel Corporation) '', inner diameter 3 mm, length 150 mm), and the amine was separated using a high performance liquid chromatography device ("LC-20AD" manufactured by Shimadzu Corporation) as a liquid chromatography device. At this time, the dl-tryptophan to be separated is dissolved in a mixed solvent of water/acetonitrile (1:1 (v/v)) containing about 5 mM hydrogen chloride to a concentration of about 0.1% w/v. , 2 μL of the resulting solution was injected into the column using an autosampler. Moreover, mobile phase 1 was sent to a column whose temperature was controlled to 30° C. at a rate of 0.43 mL/min.
A photodiode array detector ("SPD-M20A" manufactured by Shimadzu Corporation, detection wavelength 220 nm) was used as a detector, and the data acquired by the detector was analyzed using data analysis software ("LCsolution" manufactured by Shimadzu Corporation). The obtained chromatogram is shown in FIG.

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

(Separation of amine)
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 FIG. 2.

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

(Separation of amine)
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 FIG. 3.

[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)) as a solvent was prepared.

(Separation of amine)
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 FIG.

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

(Separation of amine)
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 FIG.

[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)) as a solvent was prepared.

(Separation of amine)
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 FIG.

[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)) as a solvent was prepared.

(Separation of amine)
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 FIG.

[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)) was prepared.

(Separation of amine)
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 FIG.

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

(Separation of amine)
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 FIG.

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

(Separation of amine)
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 flow rate of the mobile phase was changed to 0.25 mL/min. The obtained chromatogram is shown in FIG.

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

(Separation of amine)
Mobile phase F was used instead of mobile phase A, and the stationary phase was a chiral stationary phase in which cellulose tris (3,5-dimethylphenylcarbamate) was immobilized on a silica gel carrier through chemical bonds (“CHIRALPAK” manufactured by Daicel Corporation). Chiral separation of dl-tryptophan was performed in the same manner as in Example 1, except that the flow rate of the mobile phase was changed to 1.0 mL/min. . The obtained chromatogram is shown in FIG. 11.

Examples 1 to 5 and Comparative Examples 1 to 5 are experimental examples using a chiral stationary phase having a ligand having a crown ether-like cyclic structure. Such a stationary phase is effective for the chiral separation of primary amines, and includes primary ammonium ions in a crown ether-like cyclic structure through hydrogen bonding, and the interaction with the chiral binaphthyl structure causes chirality in the sample. It is something that recognizes the tee.

In Examples 1 to 4, the peaks of each enantiomer were separated from each other compared to 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, compared to Comparative Example 1 using a mobile phase containing perchloric acid and Comparative Example 2 using a mobile phase containing trifluoroacetic acid, It was confirmed that the retention on the stationary phase was strong and the peak separation was large. Further, in Examples 1 to 3, no peak cracking as seen in the second peak of Comparative Example 1 was observed.

Conventionally, when using a mobile phase that contains trifluoroacetic acid and also contains an organic solvent such as acetonitrile or a hexane/ethanol mixed solvent as the main solvent, the ability to retain the amine in the stationary phase is weak and the amine cannot be separated well. It is known that there are cases. On the other hand, the mobile phases of Examples 1 to 5 contain chloride ions, even though they are mainly organic solvent-based mobile phases with low water content. It was found that it could be strongly retained in the stationary phase. It was also found that high retention performance was exhibited even when the concentration of chloride ions in the mobile phase was as low as 5 mM. On the other hand, Comparative Example 5 showed separation performance comparable to that of Example 5, but the concentration of trifluoroacetic acid (approximately 67 mM) required to obtain such separation performance was higher than that of chloride in Example 5. It was found that the concentration was 13 times higher than the ion concentration (5 mM).

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 such a stationary phase is used, there are known problems such as the inability to sufficiently retain ionic substances or achieve asymmetric recognition using a mobile phase with a low water content that is mainly an organic solvent such as acetonitrile. ing. On the other hand, the results of Example 6 show that even with a mobile phase that is mainly an organic solvent and has a low water content, good retention and chiral separation of amines can be achieved by adding triethylammonium chloride. was confirmed.

High amine separation performance can be achieved by performing liquid chromatography using mobile phases according to at least some embodiments of the present disclosure. Furthermore, by selecting highly volatile (pseudo)halide ions and acids, the adverse effect on detection by a mass spectrometer is suppressed, so it is thought that application to LC-MS is also possible.
Therefore, such a mobile phase can be widely applied to analysis and purification using various types of liquid chromatography or supercritical fluid chromatography, and is expected to be used in fields such as organic chemistry, medicine, and pharmacy.

Claims (14)

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,
The solvent is one or more solvents selected from a mixed solvent of water and an organic solvent, an organic solvent, subcritical carbon dioxide, and supercritical carbon dioxide, in which the water content is more than 0% by volume and not more than 50% by volume. Yes, mobile phase. The mobile phase according to claim 1, wherein the anion does not form a salt. The mobile phase according to claim 1, wherein the anion forms a salt with a countercation. The mobile phase according to claim 3, wherein the counter cation is one or more selected from secondary ammonium ions, tertiary ammonium ions, and quaternary ammonium ions. The mobile phase according to claim 3 or 4, further comprising an acid having a pKa of -2.0 or more and 4.0 or less in water at 25°C. The mobile phase according to any one of claims 1 to 5, wherein the anion is a chloride ion. The mobile phase according to any one of claims 1 to 6, which is used in liquid chromatography or supercritical fluid chromatography using a nonionic stationary phase (excluding size exclusion type stationary phases). The mobile phase according to claim 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. comprising a separation step of separating ionizable organic compounds by liquid chromatography or supercritical fluid chromatography;
The mobile phase used in the separation step 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 has a water content of 0. One or more solvents selected from a mixed solvent of water and an organic solvent, an organic solvent, subcritical carbon dioxide, and supercritical carbon dioxide having a content of more than 50 volume% by volume,
A method for separating ionizable organic compounds, wherein the stationary phase used in the separation step is a nonionic stationary phase (excluding a size exclusion type stationary phase). The separation method according to claim 9, wherein the anion does not form a salt. The separation method according to claim 9, wherein the anion forms a salt with a countercation. The separation method according to claim 11, wherein the counter cation is one or more selected from secondary ammonium ions, tertiary ammonium ions, and quaternary ammonium ions. The separation method according to claim 11 or 12, wherein the mobile phase further contains an acid whose pKa in water at 25° C. is −2.0 or more and 4.0 or less. Any one of claims 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. Separation method described in.

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