Method for improving separation performance of stationary phase for column chromatography

Abstract

This method for improving solid phase separation performance in column chromatography comprises a separation performance improving step of bringing a solid phase into contact with an organic solvent that swells or dissolves cellulose (4-methylbenzoate), the solid phase including a crosslinked product of cellulose (4-methylbenzoate) supported on a carrier.

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 often significant differences in efficacy, toxicity, metabolism, and distribution between optical isomers. 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. However, as described above, when the physical or chemical properties of the compounds are completely identical, it is impossible to separate optical isomers by classical methods for identifying or separating compounds based on the differences in their physical properties, such as distillation utilizing the difference in boiling point, recrystallization utilizing the difference in solubility, and liquid-liquid extraction and solid-liquid extraction utilizing the difference in distribution coefficient.

[0003] 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). For this purpose, numerous 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 a polysaccharide derivative, which is a polymer material (Patent Documents 2 to 5, Non-Patent Document 2). In recent years, a method of performing optical isomer separation in the analytical field has been frequently used, such as in the high-performance liquid chromatography (HPLC) mode or the supercritical fluid chromatography (SFC) method chromatography mode, using an analytical column filled with this chiral discriminator. In addition, actual pharmaceuticals are being manufactured using a preparative column with an enlarged analytical column.

[0004] The analytical column is intended to specify the optical purity of the optical isomers, that is, the ratio and composition of each optical isomer contained in the analytical sample. When the performance of the analytical column deteriorates during the analysis of a large number (usually several hundred to several thousand) of samples, conventionally, it has generally been discarded as its lifespan when a certain performance degradation or change occurs. The lifespan of the column varies depending on the usage conditions, and it often runs out earlier than expected. Therefore, the development of a method to extend the lifespan of the column is desired.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] Recently, a method of improving column performance by passing a specific solvent through a column with deteriorated performance has also been attempted. However, it is not easy to select a solvent and an asymmetric resolving agent that can improve the separation performance. For example, in a plurality of asymmetric resolving agents with similar structures, there are many cases where a solvent capable of improving one separation performance is not effective for improving other separation performances. Furthermore, the improvement effect of the separation performance may be affected by the flow rate and the flow-through volume of the solvent. Therefore, the relationship between the asymmetric resolving agent and the conditions for improving the separation performance suitable therefor has not yet been clarified.

[0008] An object of the present disclosure is to provide a method for improving the separation performance of a stationary phase for column chromatography having a specific polysaccharide derivative as an asymmetric resolving agent.

Means for Solving the Problems

[0009] In order to solve the above problems, the present inventors have conducted intensive studies. As a result, it has been found that in a stationary phase using a cross-linked product of cellulose (4-methylbenzoate) as an asymmetric resolving agent, the separation performance of the stationary phase can be improved by bringing the stationary phase into contact with an organic solvent that swells or dissolves cellulose (4-methylbenzoate), and the above problems can be solved. That is, the gist of the present disclosure is as follows.

[0010] [1] A method for improving the separation performance of a stationary phase for column chromatography, wherein the stationary phase is a stationary phase in which a cross-linked product of cellulose (4-methylbenzoate) is supported on a carrier, A method for improving the separation performance of a stationary phase for column chromatography, comprising a step of improving separation performance by contacting the stationary phase with an organic solvent that swells or dissolves cellulose (4-methylbenzoate). [2] The method for improving the separation performance of a stationary phase for column chromatography according to [1], wherein the organic solvent is at least one of a halogenated hydrocarbon having 1 to 4 carbon atoms and an amide. [3] The halogenated hydrocarbon is at least one of dichloromethane and chloroform, The method for improving the separation performance of a stationary phase for column chromatography according to [2], wherein the amide is N,N-dimethylformamide. [4] The method for improving the separation performance of a stationary phase for column chromatography according to any one of [1] to [3], further comprising a post-treatment step of contacting the stationary phase with an alcohol having 1 to 3 carbon atoms after the separation performance improvement step. [5] The method for improving the separation performance of a stationary phase for column chromatography according to any one of [1] to [4], further comprising a pre-treatment step of contacting the stationary phase with an alcohol having 1 to 3 carbon atoms before the separation performance improvement step. [6] The method for improving the separation performance of a stationary phase for column chromatography according to [4], wherein the contact between the stationary phase and the alcohol in the post-treatment step is performed by passing the alcohol through a column filled with the stationary phase. [7] The method for improving the separation performance of a stationary phase for column chromatography according to [5], wherein the contact between the stationary phase and the alcohol in the pre-treatment step is performed by passing the alcohol through a column filled with the stationary phase. [8] The method for improving the separation performance of a stationary phase for column chromatography according to [6] or [7], wherein the flow rate (linear velocity) of the alcohol in the liquid passing is 0.01 mm / sec or more and 10 mm / sec or less. [9] The method for improving the separation performance of the stationary phase for column chromatography according to any one of [4] to [8], wherein the alcohol is at least one alcohol selected from the group consisting of methanol, ethanol, and 2-propanol.

[10] The method for improving the separation performance of the stationary phase for column chromatography according to any one of [1] to [9], wherein the contact between the stationary phase and the organic solvent in the separation performance improvement step is performed by passing the organic solvent through a column packed with the stationary phase.

[11] A method for producing a stationary phase for column chromatography, including a separation performance improvement step of bringing a treated stationary phase in which a cross-linked product of cellulose (4-methylbenzoate) is supported on a carrier into contact with an organic solvent that swells or dissolves cellulose (4-methylbenzoate), A method for producing a stationary phase for column chromatography, satisfying at least one of the following (A) and (B). (A) The separation coefficient (α value) of the stationary phase is higher than that of the treated stationary phase. (B) The number of theoretical plates (N) of the column of the stationary phase is higher than that of the treated stationary phase. [Advantages of the Invention]

[0011] According to the present disclosure, a method for improving the separation performance of a stationary phase for column chromatography having a specific polysaccharide derivative as an asymmetric selector can be provided. The problems and effects of the present disclosure are not specifically limited to those described above, and include those that are apparent to those skilled in the art from the entire specification. [Brief Description of the Drawings]

[0012]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0013] Hereinafter, specific embodiments of the present disclosure will be described with examples. However, each configuration and their combinations in each embodiment are examples, and within the scope not departing from the gist of the present disclosure, addition, omission, substitution, and other changes of the configuration are possible as appropriate. The present disclosure is not limited by the embodiments. In addition, each aspect disclosed in this specification can be combined with any other features disclosed in this specification. In this specification, “at least one of A and B” shall mean “A”, “B”, or “both A and B”.

[0014] 1 Method for Improving Separation Performance of Stationary Phase for Column Chromatography The method for improving the separation performance of the stationary phase for column chromatography according to an embodiment of the present disclosure includes a separation performance improvement step of bringing a stationary phase in which a cross-linked product of cellulose (4-methylbenzoate) is supported on a carrier into contact with an organic solvent that swells or dissolves cellulose (4-methylbenzoate). In the present embodiment, the stationary phase for improving the separation performance is used for various column chromatographies such as high performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC), and ion exchange chromatography (IEC).

[0015] 1.1 Separation Performance Improvement Step 1.1.1 Stationary Phase The stationary phase in the present embodiment has a cross-linked product of cellulose (4-methylbenzoate) as an asymmetric resolving agent, and the asymmetric resolving agent is supported on a carrier. 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. Among these, the carrier is preferably a porous inorganic carrier or a surface-porous inorganic carrier. Here, the porous carrier means a carrier in which pores are formed throughout the entire 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.

[0016] Examples of the porous organic carrier include polystyrene, poly(meth)acrylamide, poly(meth)acrylate ester, etc. Examples of the porous inorganic carrier include silica, alumina, magnesia, glass, kaolin, titanium oxide, zirconium oxide, silicate, hydroxyapatite, etc., and silica gel is preferable. Examples of the porous organic-inorganic composite carrier include an organic-inorganic composite carrier formed by a sol-gel reaction of an alkoxysilane and an alkyl-substituted or alkylene-substituted alkoxysilane compound.

[0017] The cross-linked product of cellulose (4-methylbenzoate) as an asymmetric discriminator is a product in which at least cellulose (4-methylbenzoate) molecules are polymerized and insolubilized by cross-linking. The cross-linking may be by a cross-linking agent, a cross-linking catalyst, or both. The cross-linked product of cellulose (4-methylbenzoate) is formed so as to coat the carrier. Also, the cross-linked product of cellulose (4-methylbenzoate) may be linked to the carrier via a functional group on the carrier surface or a functional group introduced onto the carrier surface by surface treatment. Further, the linking may be carried out, for example, via a cross-linking agent. Note that cellulose (4-methylbenzoate), which is a compound before cross-linking of the asymmetric resolving agent, has a number average degree of polymerization of the cellulose part (average number of pyranose rings contained in one molecule) of 5 or more, preferably 10 or more. Although there is no particular upper limit, from the viewpoint of handleability, it is preferably 1000 or less. Further, cellulose (4-methylbenzoate) is a compound in which some or all of the hydroxyl groups of cellulose are modified with 4-methylbenzoyl groups, and a crosslinkable group may be introduced into a part of the hydroxyl groups.

[0018] 1.1.2 Organic solvent The organic solvent to be brought into contact with the stationary phase is a solvent that swells or dissolves cellulose (4-methylbenzoate) (hereinafter sometimes simply referred to as "organic solvent"). Regarding the principle by which the separation performance of the stationary phase is improved by the organic solvent that swells or dissolves cellulose (4-methylbenzoate) (that is, the compound before cross-linking of the cellulose (4-methylbenzoate) cross-linked product, which is an asymmetric resolving agent), the present inventors presume as follows.

[0019] Cellulose derivatives such as cellulose (4-methylbenzoate) are optically active polymers having a one-way wound helical structure. Elements contributing to the asymmetric discrimination ability of cellulose derivatives include not only the side chain structure of cellulose (in this embodiment, the 4-methylbenzoyl group), but also the helical structure of the cellulose derivative. Therefore, as a cause of the decrease in the separation performance of the stationary phase, during the repeated introduction of the analysis sample into the column filled with the stationary phase and the passage of various mobile phases, due to changes in the helix diameter, changes in the helix angle, etc., the helical structure of the cellulose derivative cannot maintain a state suitable for optical resolution. In this embodiment, it is presumed that by bringing an organic solvent that swells or dissolves cellulose (4-methylbenzoate) into contact with the stationary phase, the degree of freedom of the helical structure of cellulose (4-methylbenzoate) increases and returns to a state suitable for optical resolution. In addition, in this embodiment, since a cross-linked product of cellulose (4-methylbenzoate) is employed as the asymmetric discriminator, even when an organic solvent that swells or dissolves cellulose (4-methylbenzoate) is brought into contact with the stationary phase, the asymmetric discriminator does not fall off from the carrier and the helical structure of cellulose (4-methylbenzoate) can be controlled.

[0020] Examples of the organic solvent include halogenated hydrocarbons having 1 to 4 carbon atoms, amides, ethers having 4 to 8 carbon atoms, ketones having 3 to 8 carbon atoms, esters having 3 to 8 carbon atoms, sulfoxides, etc. that exist as liquids under the conditions under which the separation performance improvement step is performed. Among these, the organic solvent is preferably one or both of halogenated hydrocarbons having 1 to 4 carbon atoms and amides in terms of excellent separation performance improvement effect, and more preferably a halogenated hydrocarbon having 1 to 4 carbon atoms. Hereinafter, these solvents will be described in more detail.

[0021] Hydrocarbons with 1 to 4 carbon atoms and halogenated are compounds in which the hydrogen atoms bonded to the carbon atoms of hydrocarbons with 1 to 4 carbon atoms are replaced by halogen atoms. Examples of the hydrocarbons in halogenated hydrocarbons include linear, branched or cyclic alkanes or alkenes. The hydrocarbon is preferably a linear or branched alkane, more preferably a linear alkane. The number of carbon atoms of the hydrocarbon is preferably 3 or less, more preferably 2 or less, and still more preferably 1. Further, examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc., and a chlorine atom is preferable. Specific halogenated hydrocarbons with 1 to 4 carbon atoms include difluoromethane, trichlorofluoromethane, dichloromethane, chloroform, dichloroethane, dichloropropane, dibromomethane, bromoform, diiodomethane, etc. Among these, the halogenated hydrocarbon is preferably dichloromethane or chloroform, more preferably dichloromethane.

[0022] Examples of the amide include formamide, N-alkylformamide, N,N-dialkylformamide, N-alkylacetamide, N,N-dialkylacetamide, etc. The number of carbon atoms of the alkyl group in N-alkylformamide, N,N-dialkylformamide, N-alkylacetamide or N,N-dialkylacetamide is usually 1 or more and 3 or less, preferably 1 or 2, and more preferably 1. Examples of N-alkylformamide include N-methylformamide, N-ethylformamide, N-propylformamide, etc., examples of N,N-dialkylformamide include N,N-dimethylformamide, N,N-diethylformamide, etc. Further, examples of N-alkylacetamide include N-ethylacetamide, etc., and examples of N,N-dialkylacetamide include N,N-dimethylacetamide, N,N-diethylacetamide, etc. Among these, the amide is preferably N,N-dimethylformamide.

[0023] In addition, examples of ethers having 4 to 8 carbon atoms include tetrahydrofuran, 1,4-dioxane, diethyl ether, diisopropyl ether, tert-butyl methyl ether, and the like. Examples of ketones having 3 to 8 carbon atoms include acetone, diethyl ketone, methyl ethyl ketone, methyl isobutyl ketone, and the like. Examples of esters having 3 to 8 carbon atoms include methyl acetate, ethyl acetate, methyl benzoate, and the like. Examples of sulfoxides include dimethyl sulfoxide and the like.

[0024] In addition, organic solvents such as halogenated hydrocarbons having 1 to 4 carbon atoms, amides, ethers having 4 to 8 carbon atoms, ketones having 3 to 8 carbon atoms, esters having 3 to 8 carbon atoms, and sulfoxides may be used alone or in combination of two or more.

[0025] 1.1.3 Contact Conditions The method of bringing the stationary phase into contact with the organic solvent is not particularly limited, and for example, a method of passing the organic solvent through a column filled with the stationary phase can be preferably employed. Hereinafter, the method of passing the organic solvent through the column will be described.

[0026] In this method, the organic solvent may be passed continuously or intermittently, but continuous passing is preferred. Intermittent passing of the organic solvent means that in the passing of the organic solvent, the stationary phase is immersed in the organic solvent in the column by temporarily stopping the passing, and the operation of restarting the passing after maintaining this immersion state for a certain period of time is performed one or more times.

[0027] The flow rate (linear velocity) of the organic solvent is not particularly limited as long as the column pressure can be maintained within a range equivalent to the allowable column pressure range in the analysis of the sample. For example, when analyzing the sample, if the allowable pressure of the column is 50 MPa (500 kg / cm 2In the following cases, the upper limit of the flow rate may be adjusted so that the column pressure becomes 50 MPa or less. Since the pressure inside the column depends on factors such as the particle size of the stationary phase and the viscosity of the organic solvent, the upper limit of the flow rate of the organic solvent may be appropriately determined while considering these factors. On the other hand, the lower limit of the flow rate of the organic solvent is not particularly limited and is usually more than 0 mm / sec. In addition, the flow volume of the organic solvent is usually 1.0 times or more with respect to the column volume, and from the viewpoint of obtaining a sufficient separation performance improvement effect, it is preferably 2.0 times or more, 5.0 times or more, or 10 times or more. Although there is no upper limit to the flow volume of the organic solvent, from a practical viewpoint, it is preferably 1000 times or less, more preferably 500 times or less, 100 times, or 50 times or less with respect to the column volume. The flow time of the organic solvent may be appropriately selected according to the flow rate and flow volume of the organic solvent. However, when the organic solvent is intermittently passed through the column, from the viewpoint of ensuring the separation performance improvement effect, it is preferable that the flow time of the organic solvent is 1 hour or more. In addition, the temperature of the organic solvent is not particularly limited as long as the organic solvent exists as a liquid, and it is preferably 0 °C or higher, more preferably room temperature. In this specification, room temperature means a temperature range of 20 °C or higher and 35 °C or lower. In a column chromatography apparatus equipped with a column oven, the set temperature of the column oven is taken as the temperature of the organic solvent.

[0028] 1.1.4 Evaluation Method for Separation Performance As indices for evaluating the separation performance of the stationary phase, the retention factor (k'), separation factor (α value), column theoretical plate number (N), and peak symmetry (Ps) can be used. Each index is defined as follows. · Retention factor k1' = (t1 - t0) / t0 · Retention factor k2' = (t2 - t0) / t0 t0, dead time (the time from when a substance that does not interact with the stationary phase is introduced into the column until it elutes. For convenience, the elution time of tri-tert-butylbenzene is taken as the dead time.) t1: Elution time of a component that is more weakly retained t2: Elution time of a component that is more strongly retained ·Separation factor α = k2’ / k1’ (i.e., (retention factor of the more strongly retained component) / (retention factor of the more weakly retained component)) ·Theoretical plate number N = 5.54 × (tr / W0.5) 2 tr = retention time W = peak width at the position where the peak height is 1 / 2 (half-width) ·Peak symmetry (Ps) = W(5%) / 2a W(5%): Peak width at the height of 5% of the peak height (see Fig. 1) a: Width of the rising side of the peak at the height of 5% of the peak height when the peak is bisected by a perpendicular line from the peak top (see Fig. 1)

[0029] In this embodiment, in order to evaluate that the separation performance is improved, at least one of the retention factor, separation factor, column theoretical plate number, and peak symmetry may be improved. However, it is preferable that at least one of the retention factor, separation factor, and column theoretical plate number is improved. More preferably, one or both of the separation factor and column theoretical plate number are improved. Even more preferably, both the separation factor and column theoretical plate number are improved. Particularly preferably, all are improved.

[0030] 1.2 Post-treatment process In the method for improving separation performance according to this embodiment, after the separation performance improvement step, it is preferable to perform a post-treatment step of bringing the stationary phase into contact with an alcohol having 1 to 3 carbon atoms (hereinafter sometimes simply referred to as "alcohol"). By performing such a post-treatment step, it becomes possible to further improve the separation performance. The inventors presume that the reason is that the helical structure of cellulose (4-methylbenzoate) controlled in the separation performance improvement step is gradually immobilized by contact with an alcohol having 1 to 3 carbon atoms, which is a poor solvent but has good affinity with cellulose (4-methylbenzoate).

[0031] 1.2.1 Alcohol Examples of alcohols having 1 to 3 carbon atoms include methanol, ethanol, n-propanol, 2-propanol and the like. Among these, the alcohol is preferably methanol, ethanol or 2-propanol, and more preferably ethanol. The alcohol having 1 to 3 carbon atoms may be used alone or in combination of two or more.

[0032] 1.2.2 Contact conditions The method of bringing the stationary phase into contact with the alcohol having 1 to 3 carbon atoms is not particularly limited, and for example, a method of passing an organic solvent through a column filled with the stationary phase can be preferably employed. The alcohol may be passed continuously or intermittently, but continuous passage is preferred. Note that the intermittent passage of alcohol has the same meaning as the intermittent passage of the organic solvent. The flow rate (linear velocity) of the alcohol is usually 0.01 mm / sec or more, preferably 0.05 mm / sec or more, more preferably 0.1 mm / sec or more, still more preferably 0.3 mm / sec or more, and usually 10 mm / sec or less, preferably 5 mm / sec or less, more preferably 1 mm / sec, still more preferably 0.5 mm / sec or less.

[0033] The amount of alcohol passed is usually 1.0 times or more based on the column volume, and from the viewpoint of further enhancing the separation performance improvement effect, it is preferably 2.0 times or more or 3.0 times or more. Although there is no upper limit to the amount of alcohol passed, from a practical viewpoint, it is preferably 1000 times or less, more preferably 500 times or less, 100 times or 10 times or less based on the column volume. The passage time of the alcohol may be appropriately selected according to the flow rate and the amount of alcohol passed. However, when the alcohol is passed intermittently through the column, it is preferable that the passage time of the alcohol is 1 hour or more. As the temperature of the alcohol during the passage of the alcohol, the same temperature as that of the organic solvent during the separation performance improvement step can be adopted.

[0034] 1.3 Pretreatment step In the separation performance improvement method according to this embodiment, it is preferable to perform a pretreatment step of bringing a stationary phase into contact with an alcohol having 1 to 3 carbon atoms (hereinafter sometimes simply referred to as "alcohol") before the separation performance improvement step. This is because, in the separation performance improvement step, when an organic solvent having low compatibility with the solvent (mobile phase) in the column is used, there is a risk that the mobile phase and the organic solvent may separate from each other and the separation performance improvement effect cannot be sufficiently obtained. By passing an alcohol that is compatible with both the mobile phase and the organic solvent through the column prior to the separation performance improvement step, it is considered that such a situation can be avoided. Also, in the separation performance improvement step, in order to sufficiently exhibit the separation performance improvement effect, it is preferable to remove the analysis sample, impurities, etc. remaining in the stationary phase. Therefore, it is considered desirable to wash the stationary phase with an alcohol having 1 to 3 carbon atoms prior to the separation performance improvement step.

[0035] 1.3.1 Alcohol Examples of the alcohol having 1 to 3 carbon atoms used in the pretreatment step include the same compounds as those listed as the alcohol having 1 to 3 carbon atoms used in the post-treatment step, and the preferred embodiments are also the same. Also, when both the pretreatment step and the post-treatment step are performed, the alcohol used in the pretreatment step and the alcohol used in the post-treatment step may be the same or different, but it is preferable that they are the same. That is, it is particularly preferable to pass ethanol through the column in both the pretreatment step and the post-treatment step.

[0036] 1.3.2 Contact Conditions As an explanation of the method of bringing the stationary phase into contact with the alcohol having 1 to 3 carbon atoms, the explanation in the "Contact Conditions" item of the post-treatment step is incorporated by reference. Also, the flow rate (linear velocity) of the alcohol is not particularly limited as long as the column pressure can be maintained within a range equivalent to the column pressure range allowed in the analysis of the sample. For example, the flow rate of the alcohol in the post-treatment step can be adopted.

[0037] According to the separation performance improvement method according to this embodiment, it is possible to recover the separation performance of the stationary phase for column chromatography, which has deteriorated during use, to the same level as before use (at the time of shipment). Thus, by the separation performance improvement method according to this embodiment, even a stationary phase that has conventionally been targeted for disposal can be regenerated into a stationary phase having high separation performance, so that the life of the stationary phase can be extended. In addition, if the separation performance improvement method according to this embodiment is used for a stationary phase for column chromatography immediately after production, the separation performance of which is less than the reference value, the stationary phase can be converted into a stationary phase having the separation performance at the required level as a product. Therefore, the separation performance improvement method according to this embodiment is also effective for improving the yield in the production of the stationary phase for column chromatography.

[0038] 2 Method for producing a stationary phase for column chromatography Another embodiment of the present disclosure is a method for producing a stationary phase for column chromatography, the separation performance of which is improved by the above-described separation performance improvement method as compared with before the separation performance improvement method is carried out. In other words, another embodiment of the present disclosure is a method for producing a stationary phase for column chromatography, including a separation performance improvement step of bringing a to-be-treated stationary phase in which a cross-linked product of cellulose (4-methylbenzoate) is supported on a carrier into contact with an organic solvent that swells or dissolves cellulose (4-methylbenzoate) (hereinafter, the stationary phase before the separation performance improvement step may be simply referred to as the "to-be-treated stationary phase", and the stationary phase after the separation performance improvement step may be referred to as the "produced stationary phase"). That is, the separation performance improvement step in this embodiment is synonymous with the separation performance improvement step in the above-described separation performance improvement method. In this embodiment, the to-be-treated stationary phase is a stationary phase having low separation performance and being a target for separation performance improvement. Specifically, a stationary phase whose separation performance has deteriorated due to repeated sample analysis, a stationary phase that could not obtain sufficient separation performance at the time of production due to a defect in the production process, etc. can be adopted as the to-be-treated stationary phase.

[0039] In this embodiment, it is determined that the separation performance is improved by improving at least one, preferably both, of the separation factor (α value) and the number of theoretical plates (N) of the column. More specifically, the manufacturing method according to this embodiment satisfies at least one of the following (A) and (B), preferably (A) below, and more preferably both (A) and (B).

[0040] (A) The separation factor (α value) of the produced stationary phase is higher than that of the stationary phase to be treated. (B) The number of theoretical plates (N) of the produced stationary phase is higher than that of the stationary phase to be treated.

[0041] Also, in the manufacturing method according to this embodiment, pretreatment steps and post-treatment steps similar to the above-described separation performance improvement method may be included before and after the separation performance improvement step, respectively.

Example

[0042] 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 the gist thereof is not deviated from.

[0043] As a column filled with a stationary phase in which a cross-linked product of cellulose (4-methylbenzoate) is supported on silica gel, a CHIRALPAK (registered trademark) IJ column (0.46 cmφ × 25 cm, manufactured by Daicel Corporation) was used, and the initial (at the time of shipment) separation performance of the stationary phase; the separation performance of the stationary phase that simulated the state of deterioration over time by a deterioration test; and the separation performance of the stationary phase after implementing the separation performance improvement method were evaluated, respectively.

[0044] [Evaluation of Initial Separation Performance] The CHIRALPAK IJ column in the shipped state was connected to a liquid chromatograph (Prominence (registered trademark) manufactured by Shimadzu Corporation). Optical resolution of racemic trans-stilbene oxide was performed under the analysis conditions of this liquid chromatograph, and the separation performance of the stationary phase was evaluated. The separation factor (α value), retention factor (k'), and number of theoretical plates (N) of the stationary phase are shown in Table 1, and the chromatogram is shown in Figure 2.

[0045] (Analysis conditions) Mobile phase: hexane / 2-propanol = 90 / 10 (v / v) Flow rate: 1.0 mL / min Temperature: 25 °C Detection: UV 254 nm Conditioning time: 60 minutes

[0046] [Evaluation of separation performance after degradation test] After the initial separation performance evaluation, a degradation test was conducted under the following conditions to simulate the degradation of the stationary phase. Thereafter, the optical resolution of racemic trans-stilbene oxide was carried out under the same analysis conditions as the evaluation of the initial separation performance, and the separation performance of the stationary phase was evaluated. The separation factor (α value), retention factor (k'), and the number of theoretical plates (N) of the stationary phase are shown in Table 1, and the chromatogram is shown in Figure 3.

[0047] (Degradation test conditions) Mobile phase: DMSO Flow rate: 0.5 mL / min Temperature: 25 °C Elution time: 60 minutes

[0048] [Evaluation of separation performance after separation performance improvement] After the evaluation of the separation performance after the degradation test, it was subjected to a pretreatment step, a separation performance improvement step, and a post-treatment step under the following conditions to improve the separation performance of the stationary phase. Thereafter, the optical resolution of racemic trans-stilbene oxide was carried out under the same analysis conditions as the evaluation of the initial separation performance, and the separation performance of the stationary phase was evaluated. The separation factor (α value), retention factor (k'), and the number of theoretical plates (N) of the stationary phase are shown in Table 1, and the chromatogram is shown in Figure 4.

[0049] (Conditions of the pretreatment step) Mobile phase: ethanol Flow rate: 0.5 mL / min Temperature: 25 °C Elution time: 30 minutes (Conditions of the separation performance improvement step) Mobile phase: dichloromethane Flow rate: 0.3 mL / min Temperature: 25 °C Liquid passing time: 180 minutes (Conditions of the post-treatment process) Mobile phase: Ethanol Flow rate: 0.3 mL / min Temperature: 25 °C Liquid passing time: 50 minutes

[0050]

Table 1

[0051] From Table 1, by passing dichloromethane through the column, all of the retention coefficient, separation coefficient, and number of theoretical plates of the stationary phase are improved. In particular, it is shown that the retention coefficient and separation coefficient are improved to the same level as the column's shipped state. From the above, according to the method according to the present disclosure, the separation performance of the stationary phase whose separation performance has deteriorated due to use can be improved to the same extent as before use. Therefore, even when the separation performance of the stationary phase deteriorates over time, by using the method according to the present disclosure, the separation performance of the stationary phase is improved, so that the life of the stationary phase can be extended.

Claims

1. A method for improving the separation performance of a stationary phase for column chromatography, comprising: the stationary phase being a stationary phase in which a cross-linked product of cellulose (4-methylbenzoate) is supported on a carrier; a separation performance improvement step of bringing the stationary phase into contact with an organic solvent; the organic solvent being at least one of dichloromethane and chloroform, the method for improving the separation performance of a stationary phase for column chromatography.

2. The method for improving the separation performance of a stationary phase for column chromatography according to claim 1, further comprising a post-treatment step of bringing the stationary phase into contact with an alcohol having 1 to 3 carbon atoms after the separation performance improvement step.

3. The method for improving the separation performance of a stationary phase for column chromatography according to claim 1 or 2, further comprising a pretreatment step of bringing the stationary phase into contact with an alcohol having 1 to 3 carbon atoms before the separation performance improvement step.

4. The method for improving the separation performance of a stationary phase for column chromatography according to claim 2, wherein the contact between the stationary phase and the alcohol in the post-treatment step is performed by passing the alcohol through a column filled with the stationary phase.

5. The method for improving the separation performance of a stationary phase for column chromatography according to claim 3, wherein the contact between the stationary phase and the alcohol in the pretreatment step is performed by passing the alcohol through a column filled with the stationary phase.

6. The method for improving the separation performance of a stationary phase for column chromatography according to claim 4 or 5, wherein the flow rate (linear velocity) of the alcohol in the liquid passing is 0.01 mm / sec or more and 10 mm / sec or less.

7. The alcohol is one or more alcohols selected from the group consisting of methanol, ethanol, and 2-propanol. The method for improving the separation performance of a stationary phase for column chromatography according to any one of claims 2 to 6.

8. The method for improving the separation performance of a stationary phase for column chromatography according to any one of claims 1 to 7, wherein the contact between the stationary phase and the organic solvent in the separation performance improvement step is performed by passing the organic solvent through a column filled with the stationary phase.

9. A method for producing a stationary phase for column chromatography, comprising: a separation performance improvement step of bringing a treated stationary phase in which a cross-linked product of cellulose (4-methylbenzoate) is supported on a carrier into contact with an organic solvent. The organic solvent is at least one of dichloromethane and chloroform, A method for producing a stationary phase for column chromatography that satisfies at least one of the following (A) and (B). (A) The separation coefficient (α value) of the stationary phase is higher than that of the stationary phase to be treated. (B) The number of theoretical plates (N) of the column of the stationary phase is higher than that of the column of the stationary phase to be treated.

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