Method for improving separation performance of stationary phase for column chromatography

Abstract

To provide a method for improving separation performance of a stationary phase for column chromatography including cellulose (3-chloro-4-methylphenylcarbamate) as an asymmetric identifying agent.SOLUTION: A method for improving separation performance of a stationary phase for column chromatography includes a separation performance improvement step of processing, with an organic solvent, a stationary phase in which cellulose (3-chloro-4-methylphenylcarbamate) as an asymmetric identifying agent is carried on a carrier, and the processing is the following (A) or the following (B). (A) Processing of bringing the stationary phase and dialkyl ether having 4-10 carbon atoms into contact with each other. (B) Processing of bringing the stationary phase and first alcohol having 1-4 carbon atoms into contact with each other at a temperature of 40°C or more and the boiling point of the first alcohol or less.SELECTED DRAWING: Figure 8

Description

[Technical field]

[0001] The present disclosure relates to a method for improving the separation performance of a stationary phase for column chromatography. [Background technology]

[0002] Many chemical substances, especially organic compounds, have completely identical physical or chemical properties (e.g., boiling point, melting point, solubility, etc.), but there are compounds that have different effects on living organisms, i.e., different physiological activities. A typical example is optical isomers. In terms of differences in physiological activities, especially in the pharmaceutical field, there are cases where significant differences in efficacy, toxicity, metabolism, and distribution between optical isomers are observed. Therefore, in order to ensure the safety of pharmaceuticals, the Ministry of Health, Labor and Welfare put forward the idea in the 1985 edition of the Pharmaceutical Manufacturing Guidelines that optical isomers should be considered as completely different compounds in the body. In response to this, there was a strong need in the pharmaceutical field to separate and analyze optical isomers.

[0003] However, as described above, when the physical or chemical properties are exactly the same, it is impossible to separate optical isomers by classical techniques for identifying or separating compounds, such as distillation utilizing the difference in boiling point, recrystallization utilizing the difference in solubility, liquid-liquid extraction utilizing the difference in distribution coefficient, and solid-liquid extraction.

[0004] Therefore, in order to achieve separation of optical isomers, which are extremely difficult to separate, methods have been developed in which an optically active substance different from the object of separation is allowed to act on the optical isomer as a chiral selector (asymmetry discrimination agent), and each isomer is recognized and separated based on the difference in the interaction between the optically active substance and the optical isomer (Patent Document 1, Non-Patent Document 1).

[0005] For this purpose, many chiral selectors have been investigated. Currently, the chiral selector that is most widely used in the world and can achieve the highest separation success rate is said to be a polysaccharide derivative, which is a polymeric material (Patent Documents 2 to 5, Non-Patent Document 2). In recent years, a method of separating optical isomers using an analytical column packed with this asymmetric discriminating agent in a high performance liquid chromatography (HPLC) mode, a supercritical fluid chromatography (SFC) method chromatography mode, or the like has been widely used in the analytical field. In addition, actual pharmaceuticals are manufactured using a preparative column that is a larger version of the analytical column.

[0006] The purpose of analytical columns is to determine the optical purity of optical isomers, i.e., the ratio and composition of each optical isomer contained in an analytical sample. The performance of analytical columns deteriorates as a large number of samples (usually hundreds to thousands) are analyzed, and conventionally, columns have generally been discarded when a certain level of performance degradation or change occurs. The column life varies depending on the conditions of use, and often expires earlier than expected. Therefore, there is a demand for the development of a method to extend the column life.

[0007] Recently, attempts have been made to improve column performance by passing a specific solvent through a column with deteriorated performance. For example, Patent Document 6 discloses a method of passing dichloromethane through a column packed with a stationary phase made of silica gel supported with cellulose (4-methylbenzoate). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Special Publication No. 63-056208 [Patent Document 2] Special Publication No. 04-029649 [Patent Document 3] Special Publication No. 04-030376 [Patent Document 4] Special Publication No. 63-012850 [Patent Document 5] Special Publication No. 05-004377 [Patent Document 6] International Publication No. 2021 / 210661 [Non-patent literature]

[0009] [Non-Patent Document 1] PHARM TECH JAPAN, 11, 1311 (1955) [Non-Patent Document 2] Chem. Rev. 2016, 116, 3, 1094 Summary of the Invention [Problem to be solved by the invention]

[0010] According to the method described in Patent Document 6, it is possible to improve the separation performance of a stationary phase containing cellulose (4-methylbenzoate). However, it is not easy to select a combination of a solvent and an asymmetric discrimination agent that can improve the separation performance. For example, in the case of multiple types of asymmetric discrimination agents that have similar structures, a solvent that can improve the separation performance of one is often not effective in improving the separation performance of the other. Furthermore, the flow rate or flow rate of the solvent may affect the effect of improving the separation performance. Therefore, the relationship between the asymmetric discrimination agent and the conditions for improving the separation performance that are suitable for it has not yet been clarified.

[0011] An objective of the present disclosure is to provide a method for improving the separation performance of a stationary phase for column chromatography that contains cellulose (3-chloro-4-methylphenylcarbamate) as an asymmetric discrimination agent. [Means for solving the problem]

[0012] In order to solve the above problems, the present inventors have conducted extensive research. As a result, it has been found that in a stationary phase using cellulose (3-chloro-4-methylphenylcarbamate) as an asymmetric discrimination agent, the separation performance of the stationary phase can be improved by treating the stationary phase under specific conditions, thereby solving the above problems. That is, the gist of the present disclosure is as follows.

[0013] [1] A method for improving the separation performance of a stationary phase for column chromatography, comprising the steps of: The method includes a separation performance improving step of treating a stationary phase having cellulose (3-chloro-4-methylphenylcarbamate) supported on a carrier with an organic solvent, The method for improving the separation performance of a stationary phase for column chromatography, wherein the treatment is the following (A) or (B): (A) A treatment of contacting the stationary phase with a dialkyl ether having 4 to 10 carbon atoms. (B) contacting the stationary phase with a first alcohol having 1 to 4 carbon atoms at a temperature of 40° C. or higher and the boiling point of the first alcohol. [2] The method for improving separation performance of a stationary phase for column chromatography according to [1], wherein (A) is a treatment of passing the dialkyl ether through a column packed with the stationary phase. [3] The method for improving separation performance of a stationary phase for column chromatography according to [1] or [2], wherein the dialkyl ether is methyl tert-butyl ether. [4] The method for improving separation performance of a stationary phase for column chromatography according to [1], wherein (B) is a treatment of passing the first alcohol through a column packed with the stationary phase, and then heating the column in a sealed state at a temperature of 40°C or higher and lower than the boiling point of the first alcohol. [5] The method for improving separation performance of a stationary phase for column chromatography according to [1] or [4], wherein the first alcohol is one or more selected from the group consisting of methanol, ethanol, and n-butanol. [6] The method for improving separation performance of a stationary phase for column chromatography according to [1], wherein the treatment in the separation performance improving step is (B). [7] The method for improving separation performance of a stationary phase for column chromatography according to any one of [1] to [6], further comprising, after the separation performance improving step, a post-treatment step of contacting the stationary phase with a third alcohol having 1 to 4 carbon atoms. [8] The method for improving separation performance of a stationary phase for column chromatography according to [7], wherein the contact between the stationary phase and the third alcohol in the post-treatment step is carried out by passing the third alcohol through a column packed with the stationary phase. [9] The method for improving separation performance of a stationary phase for column chromatography according to [8], wherein a flow rate (linear velocity) of the third alcohol in the passing liquid is 0.01 mm / sec or more and 10.0 mm / sec or less.

[10] The method for improving separation performance of a stationary phase for column chromatography according to any one of [7] to [9], wherein the third alcohol is one or more alcohols selected from the group consisting of methanol, ethanol, and isopropanol.

[11] The method for improving separation performance of a stationary phase for column chromatography according to any one of [1] to

[10] , further comprising a pretreatment step of contacting the stationary phase with a second alcohol having 1 to 4 carbon atoms prior to the separation performance improvement step.

[12] The method for improving separation performance of a stationary phase for column chromatography according to

[11] , wherein the contact between the stationary phase and the second alcohol in the pretreatment step is carried out by passing the second alcohol through a column packed with the stationary phase.

[13] A method for producing a stationary phase for column chromatography, comprising the steps of: The method includes a separation performance improving step of treating a stationary phase to be treated, the stationary phase being made of a carrier carrying cellulose (3-chloro-4-methylphenylcarbamate), with an organic solvent, The treatment is the following (A) or the following (B), A method for producing a stationary phase for column chromatography, which satisfies the following (a) to (c): (A) A treatment of contacting the stationary phase to be treated with a dialkyl ether having 4 to 10 carbon atoms. (B) A treatment in which the stationary phase to be treated is contacted with a first alcohol having 1 to 4 carbon atoms at a temperature of 40° C. or higher and the boiling point of the first alcohol or lower. (a) The separation factor (α) of the stationary phase whose separation performance has been improved by the separation performance improving step is higher than the separation factor of the stationary phase to be treated. (b) The column theoretical plate number (N) of the stationary phase whose separation performance has been improved by the separation performance improving step is higher than the column theoretical plate number of the stationary phase to be treated. (c) The resolution (R) of the stationary phase whose separation performance has been improved by the separation performance improving step is higher than the resolution of the stationary phase to be treated. Effect of the Invention

[0014] According to the present disclosure, there is provided a method for improving the separation performance of a stationary phase for column chromatography that contains cellulose (3-chloro-4-methylphenylcarbamate) as an asymmetric discrimination agent. It is possible. The problems and advantages of the present disclosure are not limited to those specifically described above, but include those that will become apparent to a person skilled in the art from the entire specification. [Brief description of the drawings]

[0015] [Figure 1] FIG. 11 is a schematic diagram of a peak for explaining elements related to calculation of peak symmetry. [Diagram 2] FIG. 11 is a schematic diagram of peaks for explaining elements related to calculation of resolution. [Diagram 3] 1 is a chromatogram showing the initial separation performance of the stationary phase in Example 1. [Figure 4] 1 is a chromatogram showing the separation performance of the stationary phase after a deterioration test in Example 1. [Diagram 5]1 is a chromatogram showing the separation performance of a stationary phase after a separation performance improvement test in Example 1. [Figure 6] 1 is a chromatogram showing the initial separation performance of the stationary phase in Example 2. [Figure 7] 1 is a chromatogram showing the separation performance of the stationary phase after a deterioration test in Example 2. [Figure 8] 1 is a chromatogram showing the separation performance of the stationary phase after a separation performance improvement test in Example 2. [Figure 9] 1 is a chromatogram showing the initial separation performance of the stationary phase in Comparative Example 1. [Figure 10] 1 is a chromatogram showing the separation performance of the stationary phase after a deterioration test in Comparative Example 1. [Figure 11] 1 is a chromatogram showing the separation performance of a stationary phase after a separation performance improvement test in Comparative Example 1. [Figure 12] 1 is a chromatogram showing the initial separation performance of the stationary phase in Comparative Example 2. [Figure 13] 1 is a chromatogram showing the separation performance of the stationary phase after a deterioration test in Comparative Example 2. [Figure 14] 1 is a chromatogram showing the separation performance of the stationary phase after a separation performance improvement test in Comparative Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present disclosure will be described below with reference to specific embodiments, but each configuration and combination thereof in each embodiment is merely an example, and addition, omission, substitution, and other modifications of the configuration are possible as appropriate within the scope of the gist of the present disclosure. The present disclosure is not limited to the embodiments. Additionally, each feature disclosed herein may be combined with any other feature disclosed herein.

[0017] 1. Methods for improving the separation performance of stationary phases for column chromatography A method for improving the separation performance of a stationary phase for column chromatography according to one embodiment of the present disclosure includes a separation performance improving step of treating a stationary phase having cellulose (3-chloro-4-methylphenylcarbamate) supported on a carrier with an organic solvent.

[0018] In this embodiment, the stationary phase that improves separation performance is one that is used in various types of column chromatography such as high performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC), and ion exchange chromatography (IEC).

[0019] 1.1 Separation performance improvement process In this embodiment, the separation performance improving step is a step of treating a stationary phase in which cellulose (3-chloro-4-methylphenylcarbamate) is supported on a carrier with an organic solvent. The treatment referred to here is the following (A) or the following (B) (hereinafter sometimes referred to as "treatment (A)" or "treatment (B)", respectively), and is preferably the following (B). (A) A treatment of contacting the stationary phase with a dialkyl ether having 4 to 10 carbon atoms. (B) A treatment of contacting the stationary phase with a first alcohol having 1 to 4 carbon atoms at a temperature of 40° C. or higher and the boiling point of the first alcohol.

[0020] 1.1.1 Stationary phase The stationary phase in this embodiment has cellulose (3-chloro-4-methylphenylcarbamate) as an asymmetry recognition agent, and the asymmetry recognition agent is supported on a carrier.

[0021] Examples of the carrier include a porous organic carrier, a porous inorganic carrier, a porous organic-inorganic composite carrier, a surface-porous organic carrier, a surface-porous inorganic carrier, and a surface-porous organic-inorganic hybrid carrier. Of these, the carrier is preferably a porous inorganic carrier or a surface-porous inorganic carrier. The porous carrier means a carrier in which pores are formed throughout the carrier, and the surface-porous carrier means a so-called core-shell carrier having a structure in which a non-porous core is covered with a porous layer.

[0022] Examples of the porous organic carrier include polystyrene, poly(meth)acrylamide, and poly(meth)acrylic acid ester. Examples of the porous inorganic carrier include silica, alumina, magnesia, glass, kaolin, titanium oxide, zirconium oxide, silicate, and hydroxyapatite, and silica gel is preferred. Examples of the porous organic-inorganic composite carrier include an organic-inorganic composite carrier formed by the sol-gel reaction of an alkoxysilane and an alkyl- or alkoxysilane compound substituted with an ....

[0023] The chiral recognition agent, cellulose (3-chloro-4-methylphenylcarbamate), is not particularly limited as long as some or all of the hydroxyl groups of cellulose are modified with 3-chloro-4-methylphenylcarbamoyl groups, but is preferably cellulose tris(3-chloro-4-methylphenylcarbamate).

[0024] The cellulose (3-chloro-4-methylphenylcarbamate) may be used in the form of a crosslinked product. The crosslinked product of cellulose (3-chloro-4-methylphenylcarbamate) is a product in which cellulose (3-chloro-4-methylphenylcarbamate) is polymerized and insolubilized by crosslinking with other cellulose (3-chloro-4-methylphenylcarbamate). The crosslinking may be by a crosslinking agent, a crosslinking catalyst, or both.

[0025] The crosslinked product of cellulose (3-chloro-4-methylphenylcarbamate) may be a crosslinked product of a compound in which all of the hydroxyl groups of cellulose have been modified with 3-chloro-4-methylphenylcarbamoyl groups (i.e., cellulose tris(3-chloro-4-methylphenylcarbamate)), or a crosslinked product of a compound in which some of the hydroxyl groups of cellulose have been modified with 3-chloro-4-methylphenylcarbamoyl groups and crosslinkable groups have been introduced into some of the hydroxyl groups of cellulose.

[0026] The number-average degree of polymerization (average number of pyranose rings contained in one molecule) of the cellulose moiety of cellulose (3-chloro-4-methylphenylcarbamate) (in the crosslinked product, before crosslinking) is preferably 5 or more, more preferably 10 or more, and although there is no particular upper limit, from the viewpoint of handling, it is preferably 1,000 or less. That is, preferred ranges of the number-average degree of polymerization of the cellulose moiety of cellulose (3-chloro-4-methylphenylcarbamate) include ranges of 5 or more and 1,000 or less, and ranges of 10 or more and 1,000 or less.

[0027] The cellulose (3-chloro-4-methylphenylcarbamate) is formed so as to cover the carrier. The cellulose (3-chloro-4-methylphenylcarbamate) is linked to the carrier via a functional group on the carrier surface or a functional group introduced onto the carrier surface by surface treatment. The linkage may be achieved, for example, via a linking agent, a cross-linking agent, or the like.

[0028] 1.1.2 Processing (A) Treatment (A) relates to treatment of a stationary phase using a dialkyl ether having 4 to 10 carbon atoms (hereinafter, sometimes simply referred to as "dialkyl ether") as an organic solvent. More specifically, treatment (A) is a treatment in which the stationary phase is contacted with the dialkyl ether.

[0029] Examples of dialkyl ethers include diethyl ether, methyl isobutyl ether, methyl tert-butyl ether, ethyl propyl ether, ethyl isopropyl ether, dipropyl ether, diisopropyl ether, ethyl tert-butyl ether, propyl tert-butyl ether, di-n-butyl ether, diisobutyl ether, di-n-pentyl ether, and tert-butylhexyl ether. Among these, the dialkyl ether is preferably methyl tert-butyl ether, since it can provide a high effect of improving separation performance.

[0030] The method for contacting the stationary phase with the dialkyl ether is not particularly limited, and for example, a method in which the dialkyl ether is passed through a column packed with the stationary phase can be suitably employed.

[0031] The dialkyl ether may be passed continuously or intermittently, but is preferably passed continuously. The intermittent passing of the dialkyl ether means that the stationary phase in the column is immersed in the dialkyl ether by temporarily stopping the passing of the dialkyl ether, and the immersed state is maintained for a certain period of time, after which the passing of the dialkyl ether is resumed, and this operation is carried out at least once.

[0032] The flow rate (linear velocity) of the dialkyl ether is not particularly limited as long as the column pressure can be maintained within the range equivalent to the column pressure range permitted for sample analysis. For example, if the pressure permitted for the column during sample analysis is 50 MPa (500 kg / cm 2 ) or less, the upper limit of the flow rate can be adjusted so that the column pressure is 50 MPa or less. The pressure inside the column depends on the particle size of the stationary phase, the viscosity of the dialkyl ether, etc., so the upper limit of the flow rate of the dialkyl ether can be appropriately determined while taking these factors into consideration. On the other hand, the lower limit of the flow rate of the dialkyl ether is not particularly limited, and is usually more than 0 mm / sec.

[0033] The amount of the dialkyl ether passed through is preferably 1 time or more relative to the column volume, and more preferably 2 times or more, 5 times or more, or 10 times or more in terms of obtaining a high effect of improving separation performance. There is no upper limit to the amount of the dialkyl ether passed through, but from a practical viewpoint, it is preferably 1,000 times or less relative to the column volume, more preferably 500 times or less, 100 times or less, or 50 times or less. That is, preferred ranges of the amount of the dialkyl ether passed through include, for example, 1 time or more and 1,000 times or less, 2 times or more and 500 times or less, 5 times or more and 10 times or more and 50 times or less relative to the column volume.

[0034] The time for passing the dialkyl ether through the column may be appropriately selected depending on the flow rate and amount of the dialkyl ether. However, when passing the dialkyl ether through the column intermittently, the time for passing the dialkyl ether through the column is preferably 15 minutes or more, more preferably 30 minutes or more, even more preferably 60 minutes or more, and preferably 300 minutes or less, more preferably 180 minutes or less, in terms of obtaining a high effect of improving separation performance. That is, the preferred range of the time for passing the dialkyl ether through the column intermittently includes, for example, 15 minutes or more and 300 minutes or less, 30 minutes or more and 300 minutes or less, and 60 minutes or more and 180 minutes or less.

[0035] The temperature when the stationary phase is contacted with the dialkyl ether is not particularly limited as long as the dialkyl ether is present as a liquid, and is preferably 0° C. or higher, more preferably room temperature. In this specification, room temperature means a temperature range of 15° C. to 35° C. In addition, in a column chromatograph equipped with a column oven, the set temperature of the column oven is the temperature when the stationary phase is brought into contact with the dialkyl ether.

[0036] 1.1.3 Processing (B) Treatment (B) relates to a treatment of the stationary phase using a first alcohol having 1 to 4 carbon atoms (hereinafter, sometimes simply referred to as the "first alcohol") as an organic solvent. More specifically, treatment (B) is a treatment in which the stationary phase is contacted with the first alcohol at a temperature of 40° C. or higher and the boiling point of the first alcohol or lower.

[0037] Examples of the first alcohol include methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, tert-butanol, and isobutanol. Among these, the first alcohol is preferably one or more selected from the group consisting of methanol, ethanol, and n-butanol, and more preferably methanol, in terms of obtaining a high effect of improving separation performance.

[0038] The method of contacting the stationary phase with the first alcohol at a temperature of 40° C. or higher and the boiling point of the first alcohol or lower is not particularly limited, and may be, for example, a method of passing the first alcohol through a column packed with the stationary phase, and then heating the column to a predetermined temperature while the column is sealed (Method I); a method of heating the column packed with the stationary phase while passing the first alcohol through the column (Method II); and a method of passing the first alcohol heated to a predetermined temperature through a column packed with the stationary phase (Method III). Among these, Method I is more preferable in that a high effect of improving separation performance can be obtained and the amount of the first alcohol used can be reduced.

[0039] In the methods I and II, the column is preferably heated using a column oven installed in the column chromatograph, since this facilitates temperature control. When the column is heated using a column oven, the temperature of the column oven is set to the temperature at which the stationary phase is brought into contact with the first alcohol.

[0040] In the methods I to III, the temperature when the stationary phase is contacted with the first alcohol is usually 40° C. or higher, and is preferably 45° C. or higher, more preferably 50° C. or higher, in terms of obtaining a high effect of improving separation performance. The temperature when the stationary phase is contacted with the first alcohol is lower than the boiling point of the first alcohol, and is preferably lower than a temperature that is 3° C. or lower than the boiling point of the first alcohol. That is, the preferred range of the temperature when the stationary phase is contacted with the first alcohol includes, for example, a range of 40° C. or higher and a temperature that is 3° C. or lower than the boiling point of the first alcohol, 45° C. or higher and a temperature that is 50° C. or higher and a temperature that is 50° C. or higher and a temperature that is 50° C. or lower than the boiling point of the first alcohol. To give a specific example, the preferred temperature range when methanol (boiling point 64.7° C.) is used as the first alcohol includes, for example, a range of 40° C. or higher and 64.7° C. or lower, 45° C. or higher and 61.7° C. or lower, and 50° C. or higher and 60° C. or lower. In this specification, the boiling point of the first alcohol means the boiling point at atmospheric pressure. In addition, in a column chromatograph equipped with a column oven, the set temperature of the column oven is the temperature at which the stationary phase is brought into contact with the first alcohol.

[0041] In methods I to III, the first alcohol may be passed continuously or intermittently, but is preferably passed continuously. Intermittent passing of the first alcohol refers to a method in which the stationary phase in the column is immersed in the first alcohol by temporarily stopping the passing of the first alcohol, and the passing of the first alcohol is resumed after maintaining the immersed state for a certain period of time. This means that the operation of performing the above is performed one or more times.

[0042] In Methods I to III, the flow rate (linear velocity) of the first alcohol is not particularly limited as long as the column pressure can be maintained within a range equivalent to the column pressure range permitted for sample analysis. For example, when the pressure permitted for the column during sample analysis is 50 MPa (500 kg / cm 2 ) or less, the upper limit of the flow rate may be adjusted so that the column pressure is 50 MPa or less. Since the pressure inside the column depends on the particle size of the stationary phase, the viscosity of the first alcohol, etc., the upper limit of the flow rate of the first alcohol may be appropriately determined while taking these factors into consideration. On the other hand, the lower limit of the flow rate of the first alcohol is not particularly limited and is usually more than 0 mm / sec.

[0043] In methods I to III, the amount of the first alcohol passed through the column is preferably 1-fold or more, and from the viewpoint of obtaining a sufficient effect of improving separation performance, more preferably 2-fold or more, 5-fold or more, or 10-fold or more. There is no upper limit to the amount of the first alcohol passed through the column, but from the viewpoint of practical use, the amount is preferably 1,000-fold or less, more preferably 500-fold or less, 100-fold or less, or 50-fold or less. That is, preferred ranges of the amount of the first alcohol passed through the column include, for example, 1-fold or more and 1,000-fold or less, 2-fold or more and 500-fold or less, 5-fold or more and 10-fold or more and 50-fold or less, relative to the column volume.

[0044] In method I, the time for passing the first alcohol may be appropriately selected depending on the flow rate and amount of the first alcohol. However, when the first alcohol is passed through the column intermittently, the time for passing the first alcohol is preferably 15 minutes or more, more preferably 30 minutes or more, even more preferably 60 minutes or more, and preferably 300 minutes or less, more preferably 180 minutes or less, in terms of obtaining a high effect of improving separation performance. That is, preferred ranges for the time for passing the first alcohol intermittently through the column include, for example, ranges of 15 minutes or more and 300 minutes or less, 30 minutes or more and 300 minutes or less, and 60 minutes or more and 180 minutes or less.

[0045] In method I, the heating time when the column is heated to a predetermined temperature in a sealed state is preferably 12 hours or more, more preferably 18 hours or more, and even more preferably 24 hours or more, in terms of obtaining a high effect of improving separation performance. The upper limit of the heating time is not particularly limited, but from a practical viewpoint, it is preferably 240 hours or less, more preferably 216 hours or less, even more preferably 192 hours or less, and particularly preferably 120 hours or less. That is, preferred ranges of the heating time of the column in a sealed state include, for example, 12 hours or more and 240 hours or less, 12 hours or more and 216 hours or less, 18 hours or more and 192 hours or less, and 24 hours or more and 120 hours or less.

[0046] In the methods II and III, the time for passing the first alcohol is the time during which the stationary phase and the first alcohol are in contact with each other at a predetermined temperature. Therefore, the time for passing the first alcohol can be the same as the heating time for heating the column to a predetermined temperature in a sealed state in the method I.

[0047] 1.1.4 Separation performance evaluation method The indexes for evaluating the separation performance of the stationary phase include the retention factor (k'), separation factor (α), number of theoretical columns (N), peak symmetry (Ps), and resolution (R). Each index is defined as follows:

[0048] Retention factor k1'=(t1-t0) / t0 Retention factor k2'=(t2-t0) / t0 t0: Dead time (the time from when substances that do not interact with the stationary phase are introduced into the column to when they are eluted) For convenience, the elution time of tri-tert-butylbenzene is taken as the dead time.) t1: Elution time of the weaker retained component t2: Elution time of the more strongly retained component

[0049] Separation factor α = k2' / k1' (i.e., (retention factor of the more strongly retained component) / (retention factor of the more weakly retained component))

[0050] Column theoretical plate number (N) = 5.54 × (tr / W0.5) 2 tr: retention time W: Peak width at half the peak height (half width) Peak symmetry (Ps) = W(5%) / 2a W(5%): Peak width at 5% of the peak height (see Figure 1) a: The width of the rising side of the peak at 5% of the peak height when a perpendicular line is dropped from the peak top to divide the peak in half (see Figure 1)

[0051]

number

[0052] t R1 ,t R2 :Retention time (t R1 ≦t R2 ) (See Figure 2) W1, W2: Peak width (see Figure 2) W 0.5h1 ,W 0.5h2 : Peak width at half the peak height (half width; see Figure 2)

[0053] In this specification, the separation performance is evaluated as being improved when the value of one or more of the indexes of the retention factor, separation factor, theoretical number of columns, and resolution has increased, and when none of the indexes has decreased in value. In this embodiment, it is preferable that the value of one or more of the indexes of the separation factor, theoretical number of columns, and resolution has increased, and it is preferable that the values ​​of two or more of the separation factor, theoretical number of columns, and resolution have increased, and it is preferable that the values ​​of all of the indexes of the separation factor, theoretical number of columns, and resolution have increased. In addition, since the separation performance improvement method according to this embodiment is particularly effective in improving the theoretical number of columns and resolution, it is also particularly preferable that the values ​​of the theoretical number of columns and resolution have increased.

[0054] 1.2 Post-processing In this embodiment, after the separation performance improving step, it is preferable to carry out a post-treatment step of contacting the stationary phase with a third alcohol having 1 to 4 carbon atoms (hereinafter, sometimes simply referred to as "third alcohol"). By carrying out such a post-treatment step, it becomes possible to stably improve the separation performance.

[0055] 1.2.1 Tertiary alcohol Examples of the third alcohol having 1 to 4 carbon atoms include methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, tert-butanol, and isobutanol. Of these, the third alcohol is preferably one or more selected from the group consisting of methanol, ethanol, and isopropanol, and more preferably ethanol. In addition, from the viewpoint of more stably improving the separation performance, it is also preferable that the third alcohol used in the post-treatment step is an alcohol having a larger carbon number than the first alcohol used in the treatment (B). The third alcohol having 1 to 4 carbon atoms may be used alone or in any combination and ratio of two or more.

[0056] When the treatment (B) is carried out in the separation performance improving step, the third alcohol used in the post-treatment step may be the same alcohol as the first alcohol used in the treatment (B) or may be a different alcohol.

[0057] 1.2.2 Contact conditions The method of contacting the stationary phase with the third alcohol is not particularly limited, and for example, a method of passing the third alcohol through a column packed with the stationary phase can be suitably adopted. The passing of the third alcohol may be performed continuously or intermittently, but is preferably performed continuously. The intermittent passing of the third alcohol is synonymous with the intermittent passing of the dialkyl ether in the treatment (A) of the separation performance improvement step.

[0058] The flow rate (linear velocity) of the third alcohol is preferably 0.01 mm / sec or more, more preferably 0.05 mm / sec or more, even more preferably 0.1 mm / sec or more, particularly preferably 0.3 mm / sec or more, and is preferably 10.0 mm / sec or less, more preferably 8.0 mm / sec or less, even more preferably 6.0 mm / sec or less, particularly preferably 4.0 mm / sec or less. That is, preferred ranges of the flow rate of the third alcohol include 0.01 mm / sec or more and 10.0 mm / sec or less, 0.05 mm / sec or more and 8.0 mm / sec or less, 0.1 mm / sec or more and 6.0 mm / sec or less, and 0.3 mm / sec or more and 4.0 mm / sec or less.

[0059] The amount of the third alcohol passed through is preferably 1-fold or more relative to the column volume, and from the viewpoint of further enhancing the effect of improving the separation performance, more preferably 2-fold or more or 3-fold or more. There is no upper limit to the amount of the third alcohol passed through, but from a practical viewpoint, it is preferably 1,000-fold or less relative to the column volume, more preferably 500-fold or less, 100-fold or less, or 10-fold or less. That is, preferred ranges of the amount of the third alcohol passed through include, for example, 1-fold or more and 1,000-fold or less, 1-fold or more and 500-fold or less, 2-fold or more and 100-fold or less, and 3-fold or more and 10-fold or less relative to the column volume.

[0060] The time for passing the third alcohol may be appropriately selected depending on the flow rate and amount of the third alcohol. However, when the third alcohol is passed through the column intermittently, the time for passing the third alcohol is preferably 15 minutes or more, more preferably 30 minutes or more, even more preferably 60 minutes or more, and preferably 300 minutes or less, more preferably 180 minutes or less. That is, the preferred range of the time for passing the third alcohol intermittently through the column is, for example, 15 minutes or more and 300 minutes or less, 30 minutes or more and 300 minutes or less, and 60 minutes or more and 180 minutes or less.

[0061] In the post-treatment step, it is preferable to perform the post-treatment step under conditions in which the alcohol is not intentionally heated. Therefore, the temperature of the third alcohol during the passage of the third alcohol is preferably 0° C. or higher and room temperature or lower, more preferably room temperature.

[0062] 1.3 Pretreatment process In this embodiment, it is preferable to carry out a pretreatment step of contacting the stationary phase with a second alcohol having 1 to 3 carbon atoms (hereinafter, sometimes simply referred to as "second alcohol") before the separation performance improvement step. This is because, when an organic solvent having low compatibility with the solvent (mobile phase) in the column is used in the separation performance improvement step, the mobile phase and the organic solvent are separated from each other and separated. This is believed to be because, although there is a risk that the effect of improving separation performance may not be fully obtained, such a situation can be avoided by passing an alcohol compatible with both the mobile phase and the organic solvent through the column prior to the separation performance improvement step. Also, in order to fully obtain the effect of improving separation performance in the separation performance improvement step, it is preferable to remove the analytical sample and impurities remaining in the stationary phase, and therefore it is desirable to wash the stationary phase with an alcohol compatible with both the mobile phase and the organic solvent prior to the separation performance improvement step.

[0063] 1.3.1 Secondary alcohol The second alcohol used in the pretreatment step may be the same as the compound listed as the third alcohol used in the posttreatment step, and the preferred embodiments are also the same. In addition, when both the pretreatment step and the posttreatment step are performed, the second alcohol used in the pretreatment step and the third alcohol used in the posttreatment step may be the same or different, but are preferably the same. That is, it is particularly preferable to contact the stationary phase with ethanol in both the pretreatment step and the posttreatment step.

[0064] 1.3.2 Contact conditions The method of contacting the stationary phase with the second alcohol is described in the section "1.2.2 Contact Conditions" in the post-treatment step. However, the flow rate (linear velocity) of the second alcohol may be the same as the flow rate of the third alcohol in the post-treatment step, but is not particularly limited as long as the column pressure can be maintained within the same range as the column pressure range acceptable for sample analysis.

[0065] According to the method for improving separation performance of this embodiment, the separation performance of a stationary phase for column chromatography that has deteriorated with use can be restored to the same level or higher than that before use (at the time of shipment). Thus, according to the method for improving separation performance of this embodiment, even a stationary phase that would have been discarded in the past can be regenerated into a stationary phase with high separation performance, and the life of the stationary phase can be extended.

[0066] In addition, if the method for improving separation performance according to the present embodiment is applied to a stationary phase for column chromatography immediately after production, the stationary phase having a separation performance below a reference value, the stationary phase can be converted into a stationary phase having a required level of separation performance as a product. Therefore, the method for improving separation performance according to the present embodiment is also effective in improving the yield in the production of a stationary phase for column chromatography.

[0067] 2. Method for producing stationary phase for column chromatography Another embodiment of the present disclosure is a method for producing a stationary phase for column chromatography having improved separation performance compared to before the separation performance improvement method is carried out, by the above-mentioned separation performance improvement method. In other words, another embodiment of the present disclosure is a method for producing a stationary phase for column chromatography, comprising a separation performance improvement step of treating a treated stationary phase having cellulose (3-chloro-4-methylphenylcarbamate) supported on a carrier with an organic solvent, the treatment being the following (A) or the following (B) (in this specification, the stationary phase before the separation performance improvement step is sometimes referred to as the "treated stationary phase", and the stationary phase after the separation performance improvement step is sometimes referred to as the "stationary phase with improved separation performance"). (A) A treatment in which the stationary phase to be treated is contacted with a dialkyl ether having 4 to 10 carbon atoms. (B) A treatment in which the stationary phase to be treated is contacted with a first alcohol having 1 to 4 carbon atoms at a temperature of 40° C. or higher and the boiling point of the first alcohol.

[0068] That is, the separation performance improving step in this embodiment is synonymous with the separation performance improving step in the above-mentioned separation performance improving method.

[0069] In this embodiment, the stationary phase to be treated is a stationary phase that has low separation performance and is to be improved in separation performance. Specifically, a stationary phase whose separation performance has been reduced by repeated sample analysis, a stationary phase whose separation performance is insufficient at the time of manufacture due to a defect in the manufacturing process, etc. may be used as the stationary phase to be treated.

[0070] In this embodiment, the separation performance of the stationary phase is determined to be improved by improving the separation factor (α), the number of theoretical columns (N), and the resolution (R). That is, the production method according to this embodiment satisfies the following (a) to (c). (a) The separation factor (α) of the stationary phase with improved separation performance is higher than that of the stationary phase to be treated. (b) The column theoretical plate number (N) of the stationary phase with improved separation performance is higher than the column theoretical plate number of the stationary phase to be treated. (c) The resolution (R) of the stationary phase having improved separation performance is higher than the resolution of the treated stationary phase.

[0071] Furthermore, in the production method according to this embodiment, from the viewpoint of producing a stationary phase for column chromatography with improved separation performance, the production method may also include a pretreatment step and a posttreatment step in the above-mentioned separation performance improvement method before and after the separation performance improvement step, respectively. EXAMPLES

[0072] Hereinafter, the present disclosure will be described more specifically with reference to examples. However, the present disclosure is not limited to the following examples as long as it does not deviate from the gist of the disclosure.

[0073] Example 1 A CHIRALPAK (registered trademark) IM column (0.46 cmφ×25 cm; manufactured by Daicel Corporation) was used as a column packed with a stationary phase consisting of cellulose tris(3-chloro-4-methylphenylcarbamate) supported on silica gel. The initial (shipped) separation performance of the stationary phase, the separation performance of the stationary phase simulating a state deteriorated over time through a deterioration test, and the separation performance of the stationary phase after implementation of the separation performance improvement method were each evaluated.

[0074] [Evaluation of initial separation performance] The CHIRALPAK (registered trademark) IM column before use was connected to a liquid chromatograph (Nexera (registered trademark) series; manufactured by Shimadzu Corporation). Using the liquid chromatograph under the analysis conditions described above, optical resolution of the racemic mixture of trans-stilbene oxide was performed to evaluate the separation performance of the stationary phase. The separation factor (α), retention factor (k'), number of theoretical columns (N), and resolution (R) of the stationary phase are shown in Table 1, and the chromatogram is shown in Figure 3.

[0075] (Analysis conditions) Mobile phase: hexane / 2-propanol = 90 / 10 (v / v) Flow rate: 1.0mL / min Temperature: 25℃ Detection: UV254nm Conditioning time: 60 minutes

[0076] [Evaluation of separation performance after deterioration test] After the initial separation performance evaluation, a deterioration test was carried out under the following conditions to simulate deterioration of the stationary phase. Then, the racemic mixture of trans-stilbene oxide was separated under the same analytical conditions as those for the evaluation of the initial separation performance, and the separation performance of the stationary phase was evaluated. The separation factor (α), retention factor (k'), number of theoretical columns (N), and resolution (R) of the stationary phase are shown in Table 1, and the chromatogram is shown in Figure 2. Shown in 4.

[0077] (Deterioration test conditions) Mobile phase: ethyl acetate Flow rate: 0.5mL / min Temperature: 25℃ Liquid passing time: 60 minutes

[0078] [Evaluation of separation performance after separation performance improvement test] After evaluating the separation performance after the deterioration test, a separation performance improvement step and a post-treatment step were carried out under the following conditions to improve the separation performance of the stationary phase. Then, the racemic mixture of trans-stilbene oxide was separated under the same analytical conditions as those for the evaluation of the initial separation performance, and the separation performance of the stationary phase was evaluated. The separation factor (α), retention factor (k'), number of theoretical columns (N), and resolution (R) of the stationary phase are shown in Table 1, and the chromatogram is shown in Figure 5.

[0079] (Conditions for the separation performance improvement process) Mobile phase: Methyl tert-butyl ether Flow rate: 1.0mL / min Temperature: 25℃ Liquid passing time: 60 minutes

[0080] (Post-processing conditions) Mobile phase: Ethanol Flow rate: 0.5mL / min Temperature: 25℃ Liquid passing time: 50 minutes

[0081] [Table 1]

[0082] From Table 1, it can be seen that the separation factor and the theoretical number of columns are improved by passing methyl tert-butyl ether and ethanol in sequence through a column containing a stationary phase in which cellulose tris(3-chloro-4-methylphenylcarbamate) is supported on a carrier. More specifically, it was confirmed that the separation factor of the stationary phase was improved to almost the same level as that of the stationary phase before use. Furthermore, it was confirmed that the theoretical number of columns, which had decreased to about 35% of the initial level in the deterioration test, was improved to more than 10% above the initial theoretical number of columns in the separation performance improvement test. From these facts, it can be seen that the method according to the present disclosure has an excellent effect of improving separation performance. It is clear that this is the case.

[0083] Example 2 A CHIRALPAK (registered trademark) IM column (0.46 cmφ×25 cm; manufactured by Daicel Corporation) was used as a column packed with a stationary phase consisting of cellulose tris(3-chloro-4-methylphenylcarbamate) supported on silica gel. The initial (shipped) separation performance of the stationary phase, the separation performance of the stationary phase simulating a state deteriorated over time through a deterioration test, and the separation performance of the stationary phase after implementation of the separation performance improvement method were each evaluated.

[0084] [Evaluation of initial separation performance] The initial separation performance of the stationary phase was evaluated in the same manner as in Example 1. The separation factor (α), retention factor (k′), column theoretical plate number (N), and resolution (R) of the stationary phase are shown in Table 2, and the chromatogram is shown in FIG.

[0085] [Evaluation of separation performance after deterioration test] The separation performance of the stationary phase after the deterioration test was evaluated in the same manner as in Example 1. The separation factor (α), retention factor (k′), number of theoretical columns (N), and resolution (R) of the stationary phase are shown in Table 2, and the chromatogram is shown in FIG.

[0086] [Evaluation of separation performance after separation performance improvement test] Except for changing the conditions of the separation performance improvement step and post-treatment step as shown below, the separation performance of the stationary phase after the separation performance improvement test was evaluated in the same manner as in Example 1. The separation factor (α), retention factor (k'), number of theoretical columns (N), and resolution (R) of the stationary phase are shown in Table 2, and the chromatogram is shown in Figure 8.

[0087] (Conditions for the separation performance improvement process) Mobile phase: Methanol Flow rate: 1.0mL / min Temperature: 25℃ Liquid passing time: 60 minutes Heating temperature after sealing the column: 60℃ Column sealing time: 24 hours

[0088] (Post-processing conditions) Mobile phase: Ethanol Flow rate: 0.5mL / min Temperature: 25℃ Liquid passing time: 20 minutes

[0089] [Table 2]

[0090] From Table 2, it can be seen that the separation factor of the stationary phase and the theoretical number of columns are improved by heating the stationary phase, in which cellulose tris(3-chloro-4-methylphenylcarbamate) is supported on the carrier, in contact with methanol, and then passing ethanol through the stationary phase. In particular, it was confirmed that the theoretical number of columns, which had decreased to about 85% of the initial number in the deterioration test, improved to 6% or more above the initial theoretical number of columns in the separation performance improvement test. From these facts, it can be seen that the method according to the present disclosure has an excellent effect of improving separation performance.

[0091] Comparative Example 1 A CHIRALPAK (registered trademark) IM column (0.46 cmφ×25 cm; manufactured by Daicel Corporation) was used as a column packed with a stationary phase consisting of cellulose tris(3-chloro-4-methylphenylcarbamate) supported on silica gel. The initial (shipped) separation performance of the stationary phase, the separation performance of the stationary phase simulating a state deteriorated over time through a deterioration test, and the separation performance of the stationary phase after implementation of the separation performance improvement method were each evaluated.

[0092] [Evaluation of initial separation performance] The initial separation performance of the stationary phase was evaluated in the same manner as in Example 1. The separation factor (α), retention factor (k′), column theoretical plate number (N), and resolution (R) of the stationary phase are shown in Table 3, and the chromatogram is shown in FIG.

[0093] [Evaluation of separation performance after deterioration test] Except for changing the deterioration test conditions as shown below, the separation performance of the stationary phase after the deterioration test was evaluated in the same manner as in Example 1. The separation factor (α), retention factor (k'), column theoretical plate number (N), and resolution (R) of the stationary phase are shown in Table 3, and the chromatogram is shown in Figure 10.

[0094] (Deterioration test conditions) Mobile phase: Tetrahydrofuran Flow rate: 0.5mL / min Temperature: 25℃ Liquid passing time: 60 minutes

[0095] [Evaluation of separation performance after separation performance improvement test] Except for changing the conditions of the separation performance improvement step as shown below, the separation performance of the stationary phase after the separation performance improvement test was evaluated in the same manner as in Example 1. The separation factor (α), retention factor (k'), number of theoretical columns (N), and resolution (R) of the stationary phase are shown in Table 3, and the chromatogram is shown in Figure 11.

[0096] (Conditions for the separation performance improvement process) Mobile phase: dichloromethane Flow rate: 0.3mL / min Temperature: 25℃ Liquid passing time: 180 minutes

[0097] [Table 3]

[0098] From Table 3, it can be seen that the theoretical plate number of the column does not increase even if dichloromethane and ethanol are passed through the column containing a stationary phase in which cellulose tris(3-chloro-4-methylphenylcarbamate) is supported on the carrier. In addition, it can be seen that although the separation factor and resolution increase in value in the above separation performance improvement test, the degree of improvement in separation performance is not sufficient.

[0099] Comparative Example 2 A CHIRALPAK (registered trademark) IJ column (0.46 cmφ×25 cm; manufactured by Daicel Corporation) was used as a column packed with a stationary phase consisting of cellulose tris(4-methylbenzoate) supported on silica gel. The initial (shipped) separation performance of the stationary phase, the separation performance of the stationary phase simulating a state deteriorated over time through a deterioration test, and the separation performance of the stationary phase after implementation of the separation performance improvement method were each evaluated.

[0100] [Evaluation of initial separation performance] The CHIRALPAK (registered trademark) IJ column before use was connected to a liquid chromatograph (Nexera (registered trademark) series; manufactured by Shimadzu Corporation). Using the liquid chromatograph under the analysis conditions described above, optical resolution of the racemic mixture of trans-stilbene oxide was performed to evaluate the separation performance of the stationary phase. The separation factor (α), retention factor (k'), number of theoretical columns (N), and resolution (R) of the stationary phase are shown in Table 4, and the chromatogram is shown in FIG. 12.

[0101] (Analysis conditions) Mobile phase: hexane / 2-propanol = 90 / 10 (v / v) Flow rate: 1.0mL / min Temperature: 25℃ Detection: UV254nm Conditioning time: 60 minutes

[0102] [Evaluation of separation performance after deterioration test] After the initial separation performance evaluation, a deterioration test was carried out under the following conditions to simulate deterioration of the stationary phase. Then, the racemic mixture of trans-stilbene oxide was separated under the same analytical conditions as those for the evaluation of the initial separation performance, and the separation performance of the stationary phase was evaluated. The separation factor (α), retention factor (k'), number of theoretical columns (N), and resolution (R) of the stationary phase are shown in Table 4, and the chromatogram is shown in Figure 13.

[0103] (Deterioration test conditions) Mobile phase: N,N-dimethylformamide Flow rate: 0.5mL / min Temperature: 25℃ Liquid passing time: 60 minutes

[0104] [Evaluation of separation performance after separation performance improvement test] After evaluating the separation performance after the deterioration test, a separation performance improvement step and a post-treatment step were carried out under the following conditions to improve the separation performance of the stationary phase. Then, the racemic mixture of trans-stilbene oxide was separated under the same analytical conditions as those for the evaluation of the initial separation performance, and the separation performance of the stationary phase was evaluated. The separation factor (α), retention factor (k'), number of theoretical columns (N), and resolution (R) of the stationary phase are shown in Table 4, and the chromatogram is shown in Figure 14.

[0105] (Conditions for the separation performance improvement process) Mobile phase: Methyl tert-butyl ether Flow rate: 1.0mL / min Temperature: 25℃ Liquid passing time: 60 minutes

[0106] (Post-processing conditions) Mobile phase: Ethanol Flow rate: 0.5mL / min Temperature: 25℃ Liquid passing time: 50 minutes

[0107] [Table 4]

[0108] From Table 2, when methyl tert-butyl ether and ethanol were sequentially passed through a column containing a stationary phase in which cellulose tris(4-methylbenzoate) was supported on the carrier, Although the values ​​of all the indices, i.e., retention factor, separation factor, number of theoretical columns, and resolution, increase, none of them increase to the same level as the initial separation performance, indicating that the improvement in separation performance is not sufficient.

[0109] As described above, according to the method of the present disclosure, the separation performance of a stationary phase whose separation performance has deteriorated due to use can be improved to the same or better than that before use. Therefore, even if the separation performance of the stationary phase deteriorates over time, the method of the present disclosure can improve the separation performance of the stationary phase, thereby extending the life of the stationary phase.

Claims

1. 1. A method for improving the separation performance of a stationary phase for column chromatography, comprising: a separation performance improving step of treating a stationary phase, in which cellulose (3-chloro-4-methylphenylcarbamate) is supported on a carrier, with an organic solvent; A method for improving the separation performance of a stationary phase for column chromatography, wherein the treatment is the following (A) or (B): (A) A treatment of contacting the stationary phase with a dialkyl ether having 4 to 10 carbon atoms. (B) A treatment of contacting the stationary phase with a first alcohol having 1 to 4 carbon atoms at a temperature of 40° C. or higher and the boiling point of the first alcohol.

2. 2. The method for improving the separation performance of a stationary phase for column chromatography according to claim 1, wherein (A) is a treatment of passing the dialkyl ether through a column packed with the stationary phase.

3. 3. The method for improving the separation performance of a stationary phase for column chromatography according to claim 1, wherein the dialkyl ether is methyl tert-butyl ether.

4. 2. The method for improving the separation performance of a stationary phase for column chromatography according to claim 1, wherein (B) is a treatment of passing the first alcohol through a column packed with the stationary phase, and then heating the column in a sealed state at a temperature of 40°C or higher and lower than the boiling point of the first alcohol.

5. 5. The method for improving separation performance of a stationary phase for column chromatography according to claim 1, wherein the first alcohol is at least one selected from the group consisting of methanol, ethanol, and n-butanol.

6. 2. The method for improving the separation performance of a stationary phase for column chromatography according to claim 1, wherein the treatment in the separation performance improving step is (B).

7. 2. The method for improving the separation performance of a stationary phase for column chromatography according to claim 1, further comprising, after the separation performance improving step, a post-treatment step of contacting the stationary phase with a third alcohol having 1 to 4 carbon atoms.

8. 8. The method for improving the separation performance of a stationary phase for column chromatography according to claim 7, wherein the contact of the stationary phase with the third alcohol in the post-treatment step is carried out by passing the third alcohol through a column packed with the stationary phase.

9. 9. The method for improving separation performance of a stationary phase for column chromatography according to claim 8, wherein the flow rate (linear velocity) of the third alcohol during the passing of the liquid is 0.01 mm / sec or more and 10.0 mm / sec or less.

10. The method for improving separation performance of a stationary phase for column chromatography according to any one of claims 7 to 9, wherein the third alcohol is one or more alcohols selected from the group consisting of methanol, ethanol, and isopropanol.

11. 2. The method for improving the separation performance of a stationary phase for column chromatography according to claim 1, further comprising a pretreatment step of contacting the stationary phase with a second alcohol having 1 to 4 carbon atoms prior to the separation performance improvement step.

12. 12. The method for improving the separation performance of a stationary phase for column chromatography according to claim 11, wherein the contact of the stationary phase with the second alcohol in the pretreatment step is carried out by passing the second alcohol through a column packed with the stationary phase.

13. A method for producing a stationary phase for column chromatography, comprising the steps of: a separation performance improving step of treating a stationary phase to be treated, which is a carrier carrying cellulose (3-chloro-4-methylphenylcarbamate), with an organic solvent; The treatment is the following (A) or the following (B), A method for producing a stationary phase for column chromatography, which satisfies the following (a) to (c): (A) A treatment in which the stationary phase to be treated is brought into contact with a dialkyl ether having from 4 to 10 carbon atoms. (B) A treatment in which the stationary phase to be treated is brought into contact with a first alcohol having 1 to 4 carbon atoms at a temperature of 40° C. or higher and the boiling point of the first alcohol or lower. (a) The separation factor (α) of the stationary phase whose separation performance has been improved by the separation performance improving step is higher than the separation factor of the stationary phase to be treated. (b) The number of theoretical columns (N) of the stationary phase whose separation performance has been improved by the separation performance improving step is higher than the number of theoretical columns of the stationary phase to be treated. (c) The degree of separation (R) of the stationary phase whose separation performance has been improved by the separation performance improving step is higher than the degree of separation of the stationary phase to be treated.

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