Method for determining the structure of substances in multi-component samples

Abstract

A novel method for determining the structure of a target substance contained in a multi-component sample is provided. According to the present invention, the structure of a target substance contained in a mixture of two or more substances can be determined. a method for separating a substance of interest from the mixture by supercritical fluid chromatography; and immersing the separated target substance in a crystalline sponge to prepare a sample for crystal structure analysis. and performing a crystal structure analysis of the sample for crystal structure analysis. Volatile solvents can be used as the mobile phase in supercritical fluid chromatography.

Description

[Technical Field]

[0001] The present invention relates to a method for determining the structure of substances in a multi-component sample. [Background technology]

[0002] Qualitative analysis of multi-component samples is performed in various industries, including drug discovery, food, cosmetics, and materials science. It is an essential process in research and development and product development. Analysis involves separating chemical substances from a multi-component mixture and determining the structure of the separated chemical substances. This involves identifying the separated chemicals, which is generally not easy. Methods for separating substances include gas chromatography (GC) and high performance liquid chromatography. GC has excellent separation ability, but The capillary electrode method is limited to volatile compounds that are stable to heat. Electrophoresis is limited to water-soluble ionic compounds. Since there are fewer restrictions on the separation of chemical substances in multi-component samples than in the above-mentioned methods, It is widely used.

[0003] In recent years, supercritical fluid chromatography (SCFC) has been used as a method for separating chemical substances from multi-component samples. SFC is attracting attention because it uses carbon dioxide in a supercritical fluid state as the mobile phase. Therefore, it is possible to dramatically reduce the amount of organic solvent used in the mobile phase compared to HPLC. It is attracting attention as a separation and analysis method with a low environmental impact, given the social need to realize a carbon-free society. In addition to its high resolution and high speed analysis, it is also compatible with a wide range of polar chemicals. It is possible to separate substances, from low polarity compounds such as volatile compounds that GC excels at, to compounds that can be separated by HPLC. Even highly polar compounds that are currently being analyzed can be separated (Non-Patent Documents 1 to 3). It is also excellent for separating and analyzing enantiomers when combined with a column (Non-Patent Document 4).

[0004] On the other hand, regarding the step of identifying separated chemical substances in the qualitative analysis of multi-component analysis, In the case of gas chromatography (GC), it can be analyzed by combining it with an EI-MS detector. It is possible to obtain MS spectrum information for chemical substances separated from each other, and to identify known components with high detection frequencies. For each compound, MS spectral information is compiled as a library, allowing for the identification of separated chemical substances. By comparing MS spectra with library compounds, chemical compounds can be identified. However, there are many sources of information, such as new compounds, that are not registered in the library. It is not possible to determine the absolute configuration of the enantiomers. In this case, APCI-MS or ESI-MS detectors are generally used. Due to the principle of ionization, the fragment ion generation pattern varies depending on the measurement model and conditions. Therefore, the information that can be obtained is limited, and it is not possible to identify chemical substances like with GC-EIMS. Therefore, the HPLC-NMR method, which combines NMR with the detector, has been put into practical use. It is not a costly method due to its low sensitivity and the need for deuterated solvents in the mobile phase. However, in some cases, the structure of unknown chemical substances can be estimated. Neither the combination with an EI-MS detector nor the combination with NMR has been put to practical use. Compared to HPLC, it has poorer ability to identify separated chemical substances.

[0005] Thus, there is still room for improvement in the qualitative analysis of multi-component samples containing various chemical substances. It was something to leave behind. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] J. Chromatogr. A, 2012, 1266, 143-148 [Non-patent document 2] J. Agric. Food Chem. 2015, 63(18), 4457-4463 [Non-patent document 3] J. Chromatogr. A, 2014, 1362, 270-277 [Non-patent document 4] Anal. Chim. Acta, 2014, 821, 1-33 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to provide a novel method for determining the structure of substances in multi-component samples. Let's say. [Means for solving the problem]

[0008] The present inventors have been conducting extensive research into methods for identifying target substances in multi-component samples. However, it has been reported that supercritical fluid chromatography (hereinafter simply referred to as "SFC") By combining this with the crystalline sponge (hereinafter simply referred to as "CS") method, it is possible to obtain It is possible to separate the target substances and identify the separated substances, and furthermore, it is possible to identify the target substances by MS and NMR. It is possible to determine the absolute configuration, i.e., to identify enantiomers, which is previously impossible. The present inventors have also found that an analytical scheme using a column with an inner diameter of 4.6 mm or less can be implemented. By combining the SFC and CS methods of the GC, it is possible to determine the structure of the target substance, including the absolute configuration. In addition, the mobile phase (modifier solvent and make-up solvent) of SFC By using solvents that can be used in the crystalline sponge method (including the top solvent), SF The process of separation by C and the process of structural determination by CS method can be carried out continuously online. The present inventors further found that the solvent used in the mobile phase of SFC and the The present invention is based on these findings. is.

[0009] According to the present invention, the following inventions are provided. [1] A method for determining the structure of a target substance contained in a mixture of two or more substances, comprising the steps of: ) separating the target substance from the mixture by supercritical fluid chromatography; and (B ) immersing the separated target substance in a crystalline sponge to prepare a sample for crystal structure analysis; (C) performing a crystal structure analysis of the sample for crystal structure analysis. [2] In step (A), a volatile solvent is used as the mobile phase for supercritical fluid chromatography. The method according to [1] above, wherein [3] After step (A) and before step (B), the volatile solvent is evaporated from the separated target substance. The method according to [2] above, further comprising the step of emitting the [4] Step (A), step (B) and step (C) are carried out consecutively, [1] to [3] above ] A method according to any one of the preceding claims. [5] The solvent used in the mobile phase of the supercritical fluid chromatography in step (A) is The method according to [4] above, wherein the solvent is usable for immersing the crystalline sponge. [6] Seamlessly connect the device that performs step (A) and the device that performs step (B) , carrying out step (A), step (B) and step (C), as described in [4] or [5] above. How to do it. [7] The octanol / water partition coefficient (log P ow )but The method according to any one of the above [1] to [6], wherein the β-saturation coefficient is -4.6 or more. [8] The method according to any one of [1] to [7] above, wherein the target substance is an enantiomer.

[0010] According to the method of the present invention, it is possible to obtain a high-quality sample even in a multi-component sample (especially a mixture of various polar substances) which is difficult to analyze. It is advantageous in that it can rapidly and accurately determine the structure (especially the absolute configuration) of the target substance. It is profitable. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the results (chromatogram) of SFC analysis of a racemic sample of omeprazole. [Figure 2] Figure 2 shows the results of crystal structure analysis of the crystalline sponge containing peak 1 in Figure 1 (asymmetric unit and solvent molecules omitted). [Figure 3] Figure 3 shows the results of crystal structure analysis of the crystalline sponge that incorporates peak 2 in Figure 1 (asymmetric unit and solvent molecules are omitted). [Figure 4] Figure 4 shows the guest structure of peak 1 in Figure 1 observed by the CS method. [Figure 5] Figure 5 shows the guest structure of peak 2 in Figure 1 observed by the CS method. [Figure 6] FIG. 6 shows the results (chromatogram) of SFC analysis of the racemic sample rac-(4R,5R)-3,5-dihydroxy-4-(3-methylbut-2-en-1-yl)-2-(3-methylbutanoyl)cyclopent-2-en-1-one. [Figure 7] FIG. 7 shows the results of crystal structure analysis of the crystalline sponge incorporating peak 1 in FIG. 6 (asymmetric unit and solvent molecules are omitted). [Figure 8] FIG. 8 shows the results of crystal structure analysis of the crystalline sponge that incorporates peak 2 in FIG. 6 (asymmetric unit and solvent molecules are omitted). [Figure 9] Figure 9 shows the guest structure of peak 1 in Figure 6 observed by the CS method. [Figure 10] Figure 10 shows the guest structure of peak 2 in Figure 6 observed by the CS method. [Figure 11] FIG. 11 shows the results (chromatogram) of SFC analysis of a racemic sample of trans-stilbene oxide. [Figure 12] FIG. 12 shows the results of crystal structure analysis of the crystalline sponge incorporating peak 1 in FIG. 11 (asymmetric unit and solvent molecules are omitted). [Figure 13] FIG. 13 shows the results of crystal structure analysis of the crystalline sponge incorporating peak 2 in FIG. 11 (asymmetric unit and solvent molecules are omitted). [Figure 14] FIG. 14 shows the guest structure of peak 1 in FIG. 11 observed by the CS method. [Figure 15] FIG. 15 shows the guest structure of peak 2 in FIG. 11 observed by the CS method. [Figure 16] Figure 16 shows the crystal structure of a [CuBr(btt)]-type crystalline sponge after immersion in methanol for several days (left: asymmetric units, right: packing structure). [Figure 17] FIG. 17 shows the results (chromatogram) of SFC analysis of a positional isomer mixture sample. [Figure 18] FIG. 18 shows the results of crystal structure analysis of the crystalline sponge incorporating peak 1 in FIG. 17 (asymmetric unit and solvent molecules are omitted). [Figure 19] FIG. 19 shows the results of crystal structure analysis of the crystalline sponge incorporating peak 2 in FIG. 17 (asymmetric unit and solvent molecules are omitted). [Figure 20] FIG. 20 shows the guest structure of peak 1 in FIG. 17 observed by the CS method. [Figure 21] FIG. 21 shows the guest structure of peak 2 in FIG. 17 observed by the CS method. [Figure 22]Figure 22 shows the crystal structure of a [Co2(R-man)2(bpy)3](NO3)2-type crystalline sponge after immersion in acetonitrile for several days (left: asymmetric units, right: packing structure). [Figure 23] FIG. 23 shows the results (chromatogram) of SFC analysis of a stereoisomer mixture sample. [Figure 24] Figure 24 shows the results of crystal structure analysis of the [Co2(R-man)2(bpy)3](NO3)2 type crystalline sponge incorporating peak 1 in Figure 23 (asymmetric unit and solvent molecules omitted). [Figure 25] Figure 25 shows the results of crystal structure analysis of the [Co2(R-man)2(bpy)3](NO3)2 type crystalline sponge incorporating peak 2 in Figure 23 (asymmetric unit and solvent molecules omitted). [Figure 26] Figure 26 shows the results of crystal structure analysis of the [Co2(R-man)2(bpy)3](NO3)2 type crystalline sponge incorporating peak 3 in Figure 23 (asymmetric unit and solvent molecules omitted). [Figure 27] Figure 27 shows the guest structure of peak 1 in Figure 23 observed by the CS method using a crystalline sponge of the [Co2(R-man)2(bpy)3](NO3)2 type. [Figure 28] Figure 28 shows the guest structure of peak 2 in Figure 23 observed by the CS method using a crystalline sponge of the [Co2(R-man)2(bpy)3](NO3)2 type. [Figure 29] Figure 29 shows the guest structure of peak 3 in Figure 23 observed by the CS method using a crystalline sponge of the [Co2(R-man)2(bpy)3](NO3)2 type. [Figure 30] Figure 30 shows the results of crystal structure analysis of the [Co2(S-man)2(bpy)3](NO3)2 type crystalline sponge incorporating peak 1 in Figure 23 (asymmetric unit and solvent molecules omitted). [Figure 31] Figure 31 shows the results of crystal structure analysis of the [Co2(S-man)2(bpy)3](NO3)2 type crystalline sponge incorporating peak 2 in Figure 23 (asymmetric unit and solvent molecules omitted). [Figure 32]Figure 32 shows the results of crystal structure analysis of the [Co2(S-man)2(bpy)3](NO3)2 type crystalline sponge incorporating peak 3 in Figure 23 (asymmetric unit and solvent molecules omitted). [Figure 33] Figure 33 shows the guest structure of peak 1 in Figure 23 observed by the CS method using a crystalline sponge of the [Co2(S-man)2(bpy)3](NO3)2 type. [Figure 34] Figure 34 shows the guest structure of peak 2 in Figure 23 observed by the CS method using a crystalline sponge of the [Co2(S-man)2(bpy)3](NO3)2 type. [Figure 35] Figure 35 shows the guest structure of peak 3 in Figure 23 observed by the CS method using a crystalline sponge of the [Co2(S-man)2(bpy)3](NO3)2 type. [Figure 36] FIG. 36 shows the results (chromatogram) of SFC analysis of a sample of a structural isomer mixture of volatile compounds. [Figure 37] FIG. 37 shows the results of crystal structure analysis of the crystalline sponge incorporating peak 1 in FIG. 36 (asymmetric unit and solvent molecules are omitted). [Figure 38] FIG. 38 shows the results of crystal structure analysis of the crystalline sponge incorporating peak 2 in FIG. 36 (asymmetric unit and solvent molecules are omitted). [Figure 39] FIG. 39 shows the guest structure of peak 1 in FIG. 36 observed by the CS method. [Figure 40] FIG. 40 shows the guest structure of peak 2 in FIG. 36 observed by the CS method. [Figure 41] FIG. 41 shows the results (chromatogram) of SFC analysis of a structural isomer mixture sample of volatile compounds. [Figure 42] FIG. 42 shows the results of crystal structure analysis of the crystalline sponge incorporating peak 1 in FIG. 41 (asymmetric unit and solvent molecules are omitted). [Figure 43] FIG. 43 shows the results of crystal structure analysis of the crystalline sponge incorporating peak 2 in FIG. 41 (asymmetric unit and solvent molecules are omitted). [Figure 44] FIG. 44 shows the guest structure of peak 1 in FIG. 41 observed by the CS method. [Figure 45] FIG. 45 shows the guest structure of peak 2 in FIG. 41 observed by the CS method. [Figure 46] Figure 46 shows the crystal structure of a crystalline sponge of the [Co2(R-man)2(bpy)3](NO3)2 type after immersion in isopropanol for several days (left: asymmetric units, right: packing structure). [Figure 47] FIG. 47 shows the results (chromatograms) of SFC analysis of a volatile racemic sample. [Figure 48] FIG. 48 shows the results of crystal structure analysis of the crystalline sponge incorporating peak 1 in FIG. 47 (asymmetric unit and solvent molecules are omitted). [Figure 49] FIG. 49 shows the results of crystal structure analysis of the crystalline sponge incorporating peak 2 in FIG. 47 (asymmetric unit and solvent molecules are omitted). [Figure 50] FIG. 50 shows the guest structure of peak 1 in FIG. 47 observed by the CS method. [Figure 51] FIG. 51 shows the guest structure of peak 2 in FIG. 47 observed by the CS method. Specific Description of the Invention

[0012] The present invention relates to the structure of a target substance contained in a multi-component sample, i.e., a mixture of two or more substances. The substance to be determined can be separated in step (A) and There are no particular limitations on the substance as long as it can be structurally analyzed in step (C), but organic compounds are also suitable. The main subject of structure determination is peptides and nucleic acids.

[0013] The method of the present invention comprises: (A) separating a target substance from a mixture of two or more substances by supercritical fluid chromatography; (B) separating the target substance by crystalline sponge (C (C) preparing a sample for crystal structure analysis by immersing the sample in a solution of and a step of performing crystal structure analysis of the compound.

[0014] In step (A), a substance to be subjected to structural determination is separated from the mixture using SFC, and the substance The SFC method separates volatile compounds and highly hydrophobic compounds into hydrophilic compounds. It is possible to separate substances with a wide range of polarities, from chromatographic to non-chromatographic compounds. and is performed according to procedures commonly used in SFC and high performance liquid chromatography (HPLC). The target substance can be separated or fractionated using a commercially available device. In this case, the target substance can be separated or isolated according to the operating procedures.

[0015] The mobile phase for SFC often contains carbon dioxide and compounds called modifiers. A solvent is added to adjust the separation. Organic solvents, volatile acids and volatile bases are often used. When extracting target substances, the carbon dioxide in the mobile phase is vaporized and dissolved in the modifier solvent. The make-up solvent is dissolved in the target solution and collected in a fraction collector. A separate agent is added between the time when the substance is separated in the column and collected in a fraction collector, etc. It is a solvent that is added to improve the solubility of the target substance. Even when dissolved in a modifier solvent, the modifier solvent and makeup solvent The compound may be dissolved in a mixed solvent of the above.

[0016] The mixture subjected to SFC in step (A) contains two or more substances, and The form of the substance is not particularly limited as long as it is contained. Examples of such compositions include food compositions (including beverage compositions), pharmaceutical compositions, health care compositions, and Personal care compositions, fragrances, natural products (e.g., food ingredients such as fruits, vegetables, spices, and herbs) ) and extracts thereof, organic synthetic compounds (e.g., paints, pigments, pesticides, insecticides), biological samples (e.g., blood, urine, saliva, nasal mucus, biological tissue, organs), environmental samples (e.g., river water, lake water) , seawater, soil), enzyme reaction products, etc. According to the method of the present invention, similar substances can be Since the method of the present invention can determine the structure of substances contained in a multi-component sample containing multiple species, This method is preferably applied to natural products and synthetic organic compounds that may contain multiple types of substances. In addition, the target substance in the mixture may be a highly polar substance, for example, Cetanol / water partition coefficient (log P ow ) value is -4.6 or more polar substances are considered as target substances. Here, the octanol / water partition ratio is the ratio between the two phases of octanol and water. The ratio of the concentration of a compound dissolved in the octanol phase to the concentration dissolved in water ( K ow ) and is used herein as the octanol / water partition coefficient, K ow Common use of log P, which is a number ow Use.

[0017] In step (B), the solution containing the target substance separated in step (A) is immersed in a crystalline sponge. Here, the crystalline sponge is a material with a regular pore structure. It is a single crystal having such a structure, for example, a metal organic framework (MOF), a covalent organic framework (CO F), porous organic molecular crystals (POMC), inorganic compounds such as zeolites, etc. However, if the single crystal has the property of incorporating compounds as guests into the pores, There is no limitation on the origin of the metal-organic framework. As examples, tertiary ligands containing a ligand having two or more coordinating sites and a metal ion as a central metal are Examples of such a polymeric metal complex include those having a three-dimensional network structure. "Ring structure" refers to a ligand (a ligand with two or more coordinating sites and other monodentate ligands) A network structure in which structural units formed by bonding with metal ions are repeated three-dimensionally. The single crystal of the metal-organic framework that can be used as a crystalline sponge is described in, for example, Nature 201 3, 495, 461-466, Chem. Commun. 2015, 51, 11252-11255, Science 2016, 353, 808-811 , Chem. Commun. 2016, 52, 7013-7015, Chem. Asian J. 2017, 12, 208-211, J. Am. Ch em. Soc. 2017, 139, 11341-11344, Chem 2017, 3, 281-289, Patent No. 5969616, etc. Considering the versatility of single crystal preparation, Nature 2013, 495 , 461-466, Chem. Commun. 2015, 51, 11252-11255, etc. 3(tpt)2·(solvent) a ] n A crystalline sponge of the type is preferred (where , X represents a halogen atom such as chlorine, bromine, iodine, or fluorine, and tpt is 2,4,6- It represents tri(4-pyridyl)-1,3,5-triazine, and solvent represents the solvent in the pores. represents the solvent contained therein, a represents any number equal to or greater than 0, and n represents any positive integer. To prepare a sample for crystal structure analysis, the target substance is immersed in a crystalline sponge. The method of incorporating the target substance into the pores as a guest is as far as the target substance is incorporated into the pores. For example, see IUCrJ 2016, 3, 139-151, Chem. Eur. J. 20 17, 23, 15035-15040, CrystEngComm, 2017, 19, 4528-4534, Org. Lett. 2018, 20, 353 6-3540, Science 2016, 353, 808-811, Chem. Commun. 2015, 51, 11252-11255, etc. By the above method, the target substance can be incorporated into the pores as a guest.

[0018] Depending on the type of crystalline sponge used, the structure of the crystalline sponge itself may be destroyed by the presence of water. Therefore, the sample containing the target substance separated in step (A) must be subjected to step (B). In particular, it is possible to incorporate various compounds as guests and determine the structure. Excellent performance in determining the concentration of [(ZnX2)3(tpt)2·(solvent) a ] n Type of Conclusion The crystalline structure of the crystalline sponge is destroyed in an environment containing water as a solvent, so water contamination should be avoided. As mentioned above, SFC requires the use of a volatile mobile phase in its separation procedure. The fraction containing the target substance separated in step (A) is then partially or completely freed from the volatile solvent. After partial evaporation, the sample can be immediately subjected to the step (B) of preparing a sample for crystal structure analysis. This is advantageous in that

[0019] That is, in the process of the present invention, in step (A), a volatile solvent (preferably containing no water) is used. It is preferable to use a mobile phase for SFC (a solvent with low volatility). After step (A) and before step (B), remove some or all of the volatile solvent from the separated target substance. The process may include a step of partially evaporating the target substance. By employing such a step, the method of the present invention can be carried out in a more efficient manner. is advantageous in that steps (A), (B) and (C) can be carried out consecutively. Here, "continuously" means that these operations are performed offline. In addition, the apparatus for carrying out the SFC method and the apparatus for carrying out the CS method are connected, and the steps (A) and (B) are carried out. (B) and (C) are carried out online.

[0020] In the above-mentioned continuous implementation, the solvent (modifier) ​​used in the mobile phase of the SFC in step (A) (including solvent and makeup solvent) used for soaking into the crystalline sponge in step (B) That is, the solvent in step (A) can be used in step (B). The same solvent as used in step (B) can be used, or the same solvent as used in step (C) can be used. If a different material is used, it may have a negative effect on the crystalline sponge (destruction or dissolution of the crystalline sponge, etc.). For example, a solvent that does not affect the reaction can be selected. Before step (B), prepare a vial containing the crystalline sponge soaked in the solvent. The substances separated by the SFC device are then placed in this vial together with the solvent used as the mobile phase for SFC. In this case, after step (A) and before step (B), the separated The process of partially or completely evaporating volatile solvents from the target substance can be omitted. Then, the equipment for carrying out process (A) and the equipment for carrying out process (B) are seamlessly connected, and the target This is advantageous in that the structural analysis of substances can be carried out quickly. Volatile compounds, in particular, tend to be lost by evaporation. Although the structure of a small amount of sample is difficult to analyze due to its inherent quality, the device for carrying out step (A) The apparatus for carrying out step (B) is seamlessly connected to separate the target compound from the mixture. By capturing the sample directly within the pores of the crystalline sponge, trace amounts of the sample can be removed without volatilization. This is extremely advantageous in that it allows structural analysis of the

[0021] Solvents that do not adversely affect the crystalline sponge include, for example, [(ZnCl2)3(tpt)2· (n-hexane) a ] n For the crystalline sponge type, n-hexane, cyclohexane hydrocarbons such as benzene, toluene, methyl tert-butyl ether (MT BE), ethers such as dimethoxyethane (DME), esters such as ethyl acetate, Ketones such as acetone and 2-butanone, and dioxide in supercritical, subcritical, gaseous or liquid states For the [CuBr(btt)] type crystalline sponge, the above hydrocarbons In addition to ketones, ethers, esters, ketones and carbon dioxide, alcohols such as alcohol, propanol, and isopropanol, and [Co2(R- man)2(bpy)3](NO3)2 type and [Co2(S-man)2(bpy )3](NO3)2 type crystalline sponges are esters, ketones, carbon dioxide and alcohols, as well as nitrites such as acetonitrile. Furthermore, the above solvents that do not adversely affect the crystalline sponge can be used in any combination. They may be mixed and used in combination.

[0022] In step (C), the crystal structure of the sample for crystallography prepared in step (B) is analyzed. The molecular structure of the target substance can be determined by X-ray diffraction, neutron beam Both diffraction and electron diffraction methods can be used. That is, the structural analysis of the target substance can be carried out according to a known method. For example, Steps (B) and (C) are described in International Publication No. 2014 / 038220 and IUCrJ 2016, 3, 139-1 51.

[0023] According to the method of the present invention, the structure of a target substance can be rapidly and accurately determined in a multi-component sample that is difficult to analyze. Even if the target substance is a mixture of isomers, These can be separated and their structures identified and determined. There are stereoisomers, structural isomers include positional isomers, and stereoisomers include conformational isomers. The configurational isomers include mirror image isomers (enantiomers) and It contains diastereoisomers and diastereoisomers. Active ingredients or compounds that are candidates for active ingredients, taste and aroma compounds, etc. are one of the enantiomers. Since only one component may function as an active ingredient, the method of the present invention can be used to detect This is highly advantageous because it allows rapid and accurate determination of the absolute configuration of compounds. [Example]

[0024] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples. It's not something like that.

[0025] Example 1: Enantiomer separation of a racemic sample by SFC (1) In Example 1, a preparative SFC system suitable for use with a column with an inner diameter of 10 mm or more was used. The semi-solid sample was subjected to enantiomeric separation. (1) Method The racemic sample was an active ingredient in a commercially available drug and available as a general reagent. Meprazole (Tokyo Chemical Industry Co., Ltd.) was used. Omeprazole (racemic) was dissolved in methanol. The solution was prepared at 10 mg / mL and subjected to supercritical fluid chromatography (SFC) under the following conditions: The samples were subjected to a chromatographic analysis using a Supercritical Fluid Chromatography System (Waters).

[0026] [Table 1]

[0027] (2) Results The chromatogram obtained under the above conditions is shown in Figure 1. The two enantiomers of benzophenone were separated within 5 minutes with a high resolution of 10.5. .

[0028] Example 2: Absolute stereostructure determination by CS method (1) In Example 2, the two enantiomers of the racemic compound separated and fractionated in Example 1 were analyzed using the CS method. The absolute three-dimensional structure was determined.

[0029] (1) Method The components of the two peaks (peaks 1 and 2) shown in Figure 1 of Example 1 were collected, and a portion (40 μ g) was transferred to a 1.2 mL V-bottom vial and the eluent, methanol (MeOH), was added under nitrogen. The mixture was evaporated under a stream of pure air. Then, one crystalline sponge (100 μm × 100 μm × 100 μm) was placed on the 100 μm and density 1.3 g / cm 3 The theoretical amount when methyl- tert-butyl ether (MTBE) and dimethoxyethane (DME) (volume ratio 9:1) The vial was capped and incubated at 50°C for 1 day. After that, the crystalline sponge was removed from the vial and analyzed by a single crystal X-ray diffractometer (Cu Kα λ=1. 5418 Å).

[0030] The crystalline sponge was prepared according to the method described in Chem. Eur. J. 2017, 23, 15035-15040. [(ZnCl2)3(tpt)2·(n-hexane) a ] n type was used. The crystalline sponge was stored soaked in n-hexane until use. The solvent was replaced with the above MTBE / DME mixed solvent before use in the experiment.

[0031] The measurement data was analyzed according to the method described in Chem. Eur. J. 2017, 23, 15035-15040.

[0032] (2) Results Measurements using a single crystal X-ray diffractometer and analysis of the measurement data revealed that the The structure of the guest compound was observed. Table 2 shows the crystal data obtained by analyzing the crystalline sponge into which the eluted compound was incorporated.

[0033] [Table 2]

[0034] When analyzing crystalline sponge incorporating compounds eluted as peaks 1 and 2, The crystal structures of the asymmetric units are shown in Figures 2 and 3, respectively. The molecules enclosed by the dotted lines are the crystallographic The guest structures observed are shown in Figures 4 and 5, respectively. These results are shown in Figure 5. From these results, the enantiomer of peak 1 is the S-isomer of omeprazole, Compound 2 was determined and identified as the R-isomer of omeprazole.

[0035] Example 3: Enantiomer separation of a racemic sample by SFC (2) In Example 3, an analytical-scale SFC instrument (SFC) suitable for use with a column with an internal diameter of 4.6 mm or less was used. Himadzu Nexera UC (Shimadzu Corporation) with a fraction collector (FR C-40 SF (Shimadzu Corporation) was used in combination to separate the enantiomers of the racemic sample. Ta.

[0036] (1) Method As a racemic natural product sample, the method described in J. Inst. Brew. 1990, 96, 137-141 was used. rac-(4R,5R)-3,5-dihydroxy-4-(3-methylbut-2-en cyclopent-2-en-1-one (ra c-(4R,5R)-3,5-dihydroxy-4-(3-methylbut-2-en-1-yl)-2-(3-methylbutanoyl)cyclopent- 2-en-1-one) (This compound undergoes tautomerization to give rac-(4R,5R)-3,4-dihydro Roxy-5-(3-methylbut-2-en-1-yl)-2-(3-methylbutanoyl) Cyclopent-2-en-1-one (rac-(4R,5R)-3,4-dihydroxy-5-(3-methylbut-2-en- We prepared a compound that can take the structure (1-yl)-2-(3-methylbutanoyl)cyclopent-2-en-1-one). The racemic compound was adjusted to 86 mg / mL with methanol and subjected to SFC under the conditions shown in Table 3. The SFC device used was a Shimadzu Nexera UC (Shimadzu Corporation). The compounds were collected using a fraction collector FRC-40 SF (Shimadzu Corporation). A constant flow of make-up solvent was introduced into the fraction collector to increase sample recovery efficiency. was added in quantity.

[0037] [Table 3]

[0038] (2) Results The chromatogram obtained under the above conditions is shown in Figure 6. The two enantiomers of the compound could be separated within 10 minutes with a resolution of 2.0.

[0039] Example 4: Absolute stereostructure determination by CS method (2) In Example 4, the two enantiomers of the racemic compound separated and fractionated in Example 3 were analyzed using the CS method. The absolute three-dimensional structure was determined.

[0040] (1) Method The components of the two peaks (peaks 1 and 2) shown in Figure 6 of Example 3 were collected and a portion (1 Transfer the aliquot (equivalent to 5 μg, approximately 1 / 40 of the total amount) to a 1.2 mL V-bottom vial and add the eluate. A mixture of mobile phase B and makeup solvent (methanol, acetonitrile, trifluoromethyl ... The solution (composed of acetic acid) was evaporated under a nitrogen stream. 0μm×100μm×100μm, and the density is 1.3g / cm 3 Theoretical amount when: 1 The vial was capped and a syringe needle was inserted into the cap. The vial was pierced and incubated at 50°C to allow the solvent to slowly evaporate. The crystal sponge was taken out and measured using a single crystal X-ray diffractometer (Cu Kα λ=1.5418Å). Ta.

[0041] The crystalline sponge was prepared according to the method described in Chem. Eur. J. 2017, 23, 15035-15040. [(ZnCl2)3(tpt)2·(n-hexane) a ] n type was used. The crystalline sponge was stored soaked in n-hexane until use.

[0042] The measurement data was analyzed according to the method described in Chem. Eur. J. 2017, 23, 15035-15040.

[0043] (2) Results Measurements using a single crystal X-ray diffractometer and analysis of the measurement data revealed that the The structure of the guest compound was observed. Table 4 shows the crystallographic data obtained when analyzing the crystalline sponge into which the eluted compound was incorporated.

[0044] [Table 4]

[0045] When analyzing crystalline sponge incorporating compounds eluted as peaks 1 and 2, The crystal structures of the asymmetric units are shown in Figures 7 and 8, respectively. The molecules enclosed by the dotted lines are the crystallographic The guest structures observed are shown in Figures 9 and 10, respectively. The results are shown in Figure 10. From these results, it can be seen that the enantiomer of peak 1 is (4S,5S)-3,5-dimer. Hydroxy-4-(3-methylbut-2-en-1-yl)-2-(3-methylbutanoyl) (4S,5S)-3,5-dihydroxy-4-(3-methylbut-2-en-1-one) n-1-yl)-2-(3-methylbutanoyl)cyclopent-2-en-1-one), and the enantiomer of peak 2 is (4R ,5R)-3,5-dihydroxy-4-(3-methylbut-2-en-1-yl)-2- (3-methylbutanoyl)cyclopent-2-en-1-one)((4R,5R)-3,5-dihydrox y-4-(3-methylbut-2-en-1-yl)-2-(3-methylbutanoyl)cyclopent-2-en-1-one), From the results of Examples 3 and 4, it was possible to determine the Even in such a case, that is, even if the amount of purified target substance is on the order of several tens of μg, the method according to the present invention can be used. It was confirmed that the structure of the target substance can be determined by this method.

[0046] Example 5: Enantiomer separation of a racemic sample by SFC (3) In Example 5, the same SFC equipment and fraction collector as in Example 3 were used, and the separation solvent was In this study, the enantiomers of a racemic sample were separated using the same solvent as that used in the CS method. Ta.

[0047] (1) Method As a racemic sample, trans-stilbene oxide is available as a general reagent. The racemic mixture of trans-stilbene oxide (Tokyo Chemical Industry Co., Ltd.) was used. ) was prepared at 10 mg / mL with methyl tert-butyl ether (MTBE), and the As in Example 3, the SFC apparatus was a Shimadzu Nexera The separated compounds were collected using a fraction collector FRC-40 (Shimadzu Corporation). The fractions were collected using a fractionator (Shimadzu Corporation). Make-up solvent was added to the collector at a constant flow rate.

[0048] [Table 5]

[0049] The chromatogram obtained under the above conditions is shown in Figure 11. The two enantiomers of the compound could be separated within 3 min with a resolution of 2.4.

[0050] Example 6: Absolute stereostructure determination by CS method (3) In Example 6, the two enantiomers of the racemic compound separated and fractionated in Example 5 were analyzed using the CS method. The absolute three-dimensional structure was determined.

[0051] (1) Method [(ZnCl2 )3(tpt)2·(n-hexane) a ] n 1 grain of crystalline sponge (size 1 00μm×100μm×100μm, density 1.3g / cm 3 The theoretical amount is: 1.3 μg) was transferred to a V-bottom 1.2 mL vial with n-hexane. The solution was removed, and 50 μL of methyl tert-butyl ether (MTBE) was added to form crystals. The solvent in the vial was replaced with MTBE. It was set to the .

[0052] The components of the two peaks (peaks 1 and 2) shown in Figure 11 of Example 5 were extracted from the above-mentioned crystalline sponge. The solution was directly dispensed into vials containing 10 μg of each peak. After the solvent has evaporated, the vial is capped, a syringe needle is inserted into the cap, and the vial is incubated at 50°C. After one day, the crystalline sponge was removed from the vial and Measurements were made using a single crystal X-ray diffractometer (Cu Kα λ=1.5418 Å).

[0053] The measurement data was analyzed according to the method described in Chem. Eur. J. 2017, 23, 15035-15040.

[0054] (2) Results Measurements using a single crystal X-ray diffractometer and analysis of the measurement data revealed that the The structure of the guest compound was observed. Table 6 shows the crystal data obtained by analyzing the crystalline sponge containing the compound eluted by the crystalline sponge. .

[0055] [Table 6]

[0056] When analyzing crystalline sponge incorporating compounds eluted as peaks 1 and 2, The crystal structures of the asymmetric units are shown in Figures 12 and 13, respectively. The molecules enclosed by dotted lines are the crystal structures. The guest structures observed are shown in Figure 14. and Fig. 15. From these results, it was found that the enantiomer of peak 1 was trans-stillate. RR isomer of benzoxide ((2R,3R)-2,3-diphenyloxirane), peak 2 is the SS form of trans-stilbene oxide ((2S,3S)-2,3-diphenyl From the results of Examples 5 and 6, it was possible to determine and identify the crystalline sponge. The compounds separated by SFC were directly collected in the vial and incorporated into the crystalline sponge. It was confirmed that structural analysis is possible.

[0057] Example 7: Preparation of methanol-resistant crystalline sponge When methanol is used as the separation solvent for SFC, [(ZnCl2)3(tpt )2·(n-hexane) a ] n The crystalline sponge type is not resistant to methanol and is SFC Therefore, in Example 7, the CS method was not able to be seamlessly implemented. We investigated the preparation of a resistant crystalline sponge.

[0058] (1) Method Chem. Asian J. 2017, 12, 208-211, [CuBr(btt)] was synthesized from triisopropyltriisonicotinic acid (btt) and CuBr. This crystalline sponge was soaked in methanol for several days, and then The crystal structure of was analyzed.

[0059] (2) Results Chem. Asian J. 2017, 12, 208-211, [CuBr(btt)] type crystalline spon It has been reported that immersion in methanol causes cracking, making it difficult to obtain good diffraction suitable for analysis. However, as a result of intensive investigation by the present inventors, it was found that the [CuBr (btt)] type crystalline sponge was surprisingly stable after being immersed in methanol for several days. We found that it is possible to measure and observe the crystal structure with high accuracy even in this case (Figure 16).

[0060] Example 8: Separation of a positional isomer mixture sample by SFC In Example 8, the same SFC equipment and fraction collector as in Example 3 were used, and the separation solvent was The same solvent as that used in the CS method was used to separate the positional isomer mixture sample. .

[0061] (1) Method As a positional isomer mixture sample, 1-acetylnaphthalene (Tokyo Chemical Industry) and 2 -acetylnaphthalene (Tokyo Chemical Industry Co., Ltd.) was used, and each compound was dissolved in methanol (MeOH) at 2 A solution containing 0 mg / mL of SF was prepared and subjected to SFC under the conditions shown in Table 7. The C device was a Shimadzu Nexera UC (Shimadzu Corporation), and the separated compounds were The product was collected using a fraction collector FRC-40 SF (Shimadzu Corporation). To improve the recovery efficiency of the fraction, a constant flow of makeup solvent was introduced into the fraction collector. Added.

[0062] [Table 7]

[0063] (2) Results The chromatogram obtained under the above conditions is shown in Figure 17. The positional isomer mixture sample (two positional isomer compounds) was separated within 6 minutes. came.

[0064] Example 9: Determination of positional isomer structure by CS method In Example 9, the two positional isomer compounds separated and isolated in Example 8 were compared with the results prepared in Example 7. The structure was determined by the CS method using a crystalline sponge.

[0065] (1) Method One grain of [CuBr(btt)] type crystalline sponge (size 100 μm) prepared in Example 7 m × 100 μm × 100 μm, and the density is 1.5 g / cm 3 Theoretical amount when: 1.5 (μg, stored in chloroform until use) was placed in a 1.2 mL V-bottom vial with chloroform and The chloroform was removed, and 50 μL of methanol was added. The solvent was replaced with methanol. Several vials were prepared and placed in a fraction collector. I did.

[0066] The components of the two peaks (peaks 1 and 2) shown in Figure 17 of Example 8 were extracted from the above-mentioned crystalline sponge. The solution was directly dispensed into vials containing approximately 100 μg of each peak. After the solvent was evaporated, the vial was capped and further incubated at 50°C for 4 days. After 3 days, the crystalline sponge was removed from the vial and analyzed by single crystal X-ray diffraction (Cu Kα λ=1 The measurement was performed at 0.5418Å.

[0067] The measurement data are published in Chem. Eur. J. 2017, 23, 15035-15040 and Chem. Asian J. 2017, 12, The analysis was carried out according to the method described in 208-211.

[0068] (2) Results Measurements using a single crystal X-ray diffractometer and analysis of the measurement data revealed that the The structure of the guest compound was observed. Table 8 shows the crystal data obtained by analyzing the crystalline sponge containing the compound eluted by the crystalline sponge. .

[0069] [Table 8]

[0070] When analyzing crystalline sponge incorporating compounds eluted as peaks 1 and 2, The crystal structures of the asymmetric units are shown in Figures 18 and 19, respectively. The molecules enclosed by dotted lines are the crystal structures. The guest structures observed are shown in Figure 20. and Fig. 21. From these results, it is clear that the positional isomer of peak 1 is 1-acetylnaphthalene. The positional isomer of peak 1 was determined and identified as 2-acetylnaphthalene. From the results of Examples 8 and 9, by using a crystalline sponge that is resistant to methanol, Compounds separated by SFC (containing methanol in the mobile phase) were directly transferred to a vial containing sponge. It was confirmed that it was possible to recover the crystals, incorporate them into a crystalline sponge, and analyze their structure.

[0071] Example 10: Preparation of acetonitrile-resistant crystalline sponge When acetonitrile is used as the separation solvent for SFC, [(ZnCl2)3(t pt)2·(n-hexane) a ] n The crystalline sponge type is not resistant to acetonitrile. Therefore, in Example 10, the SFC and CS methods were not able to be performed seamlessly. The preparation of acetonitrile-resistant crystalline sponge was investigated.

[0072] (1) Method 4,4'-bipyridine according to the method described in J. Am. Chem. Soc. 2015, 137, 12045-12049 (S)-mandelic acid (S-man) or (R)-mandelic acid (Rm an) and [Co2(R-man)2(bpy)3](N O3)2-type crystalline sponge and [Co2(S-man)2(bpy)3](NO3 Two types of crystalline sponges were prepared. These crystalline sponges were left in acetonitrile for several days. After immersion, the crystal structure was analyzed.

[0073] (2) Results [Co2(R-man)2(bpy)3](NO3)2 type crystalline sponge and [ The crystalline sponge of the Co2(S-man)2(bpy)3](NO3)2 type is Even after immersion in tritium for several days, the crystal structure can be measured and observed with high accuracy. We found that (Figure 22).

[0074] Example 11: Separation of a stereoisomeric mixture sample by SFC In Example 11, the same SFC equipment and fraction collector as in Example 3 were used, and the separation solvent The same solvent as that used in the CS method was used to separate the stereoisomer mixture sample. Ta.

[0075] (1) Method As a stereoisomer mixture sample, a rac-hydrobenzoxazole mixture of equal amounts of RR and SS isomers was used. Acetaminophen (Tokyo Chemical Industry Co., Ltd.) and meso-hydrobenzoin (Tokyo Chemical Industry Co., Ltd.) were used. 20 mg / mL (racemic) and 10 mg MeCN, respectively A solution containing 1000 mg of HCl / mL was prepared and subjected to SFC under the conditions shown in Table 9. The Shimadzu Nexera UC (Shimadzu Corporation) was used, and the separated compounds were The fractions were collected using a fraction collector FRC-40 SF (Shimadzu Corporation). To improve collection efficiency, a make-up solvent was added to the fraction collector at a constant flow rate. Ta.

[0076] [Table 9]

[0077] (2) Results The chromatogram obtained under the above conditions is shown in Figure 23. A stereoisomeric mixture sample (three stereoisomeric compounds) can be separated within 5XX minutes. Done.

[0078] Example 12: Determination of stereoisomer structure by CS method (1) In Example 12, the three stereoisomeric compounds separated and isolated in Example 11 were used to prepare the compounds in Example 10. Using the [Co2(R-man)2(bpy)3](NO3)2 type crystalline sponge The structure was determined using the CS method.

[0079] (1) Method [Co2(R-man)2(bpy)3](NO3)2 type crystals prepared in Example 10 One sponge (size 100 μm × 100 μm × 100 μm, density 1.4 g / cm m 3 The theoretical amount when calculated is 1.4 μg, stored in chloroform until use) into a V-bottom 1. The mixture was transferred to a 2 mL vial together with chloroform. The chloroform was removed and acetonitrile was added. 50 μL of acetonitrile was added to the vial, and the solvent in the crystalline sponge was replaced with acetonitrile. Several tubes were prepared and placed in a fraction collector.

[0080] The components of the three peaks (peaks 1, 2 and 3) shown in Figure 23 of Example 11 were determined by the above-mentioned crystal structure. The mixture was directly dispensed into vials containing sponge (approximately 100 μg per peak). After the solvent was evaporated to 50°C, the vial was capped and further incubated at 50°C for 3 days. After 3 days, the crystalline sponge was removed from the vial and analyzed by a single crystal X-ray diffractometer (Cu Kα λ = 1.5418 Å).

[0081] The measurement data are published in Chem. Eur. J. 2017, 23, 15035-15040 and J. Am. Chem. Soc. 2015, 1 37, 12045-12049.

[0082] (2) Results Measurements using a single crystal X-ray diffractometer and analysis of the measurement data revealed that the The structure of the guest compound was observed. Crystal data obtained by analyzing a crystalline sponge incorporating the compound eluted as peak 3 is shown in Table 10.

[0083] [Table 10]

[0084] Crystalline sponges loaded with compounds eluting as peaks 1, 2, and 3 were analyzed. The crystal structures of the asymmetric unit are shown in Figures 24, 25, and 26, respectively. The molecules are the guests incorporated into the crystalline sponge. These results are shown in Figures 27, 28, and 29, respectively. The stereoisomer in peak 1 is (S,S)-hydrobenzoin, and the stereoisomer in peak 2 is meso-hydrobenzoin The stereoisomer of peak 3 was determined and identified as (R,R)-hydrobenzoin. From the results of Examples 11 and 12, it was found that the use of crystalline sponge, which is resistant to acetonitrile, The crystalline sponge was placed in a vial and separated by SFC (with acetonitrile in the mobile phase). It is possible to directly recover the compound, incorporate it into a crystalline sponge, and analyze its structure. It was confirmed that:

[0085] Example 13: Determination of stereoisomer structure by CS method (2) In Example 13, the three stereoisomeric compounds separated and isolated in Example 11 were used to prepare the compounds in Example 10. Using the [Co2(S-man)2(bpy)3](NO3)2 type crystalline sponge The structure was determined using the CS method. (1) Method The crystal structure of the [Co2(R-man)2(bpy)3](NO3)2 type used in Example 12 Sponge was converted to its enantiomer, [Co2(S-man)2(bpy)3](NO3)2. The analysis was carried out in the same manner as in Example 12, except that the crystalline sponge was changed to a different type.

[0086] (2) Results Measurements using a single crystal X-ray diffractometer and analysis of the measurement data revealed that the The structure of the guest compound was observed. Crystal data obtained by analyzing a crystalline sponge incorporating the compound eluted as peak 3 is shown in Table 11.

[0087] [Table 11]

[0088] Crystalline sponges loaded with compounds eluting as peaks 1, 2, and 3 were analyzed. The crystal structures of the asymmetric units are shown in Figures 30, 31, and 32, respectively. The molecules are the guests incorporated into the crystalline sponge. These results are shown in Figures 33, 34, and 35, respectively. The stereoisomer in peak 1 is (S,S)-hydrobenzoin, and the stereoisomer in peak 2 is meso-hydrobenzoin. The stereoisomer of peak 3 was determined and identified as (R,R)-hydrobenzoin. From the results of Examples 11 and 13, it was found that the acetonitrile-resistant crystalline sponge was used. By doing so, the crystalline sponge was separated by SFC (with acetonitrile as the mobile phase) in a vial. The released compounds can be directly recovered, incorporated into crystalline sponges, and subjected to structural analysis. It was confirmed that...

[0089] In addition, the [Co2(R-man)2(bpy)3](NO3)2 type crystalline sponge The crystal structure incorporating peak 1 and [Co2(S-man)2(bpy)3](NO3 The crystal structures of the two types of crystalline sponges incorporating peak 3 are mirror images. The peaks were observed in the [Co2(R-man)2(bpy)3](NO3)2 type crystalline sponges. The crystal structure of [Co2(S-man)2(bpy)3](NO3)2 is shown. The crystal structure of the crystalline sponge containing peak 1 is a mirror image. Peak 2 in the crystalline sponge of the Co2(R-man)2(bpy)3](NO3)2 type The crystal structure of the incorporated compound and the [Co2(S-man)2(bpy)3](NO3)2 type The crystal structure in which peak 2 is incorporated into the crystalline sponge is also a mirror image.

[0090] Example 14: Separation of Volatile Structural Isomer Mixture Samples by SFC (1) In Example 14, the same SFC equipment and fraction collector as in Example 3 were used, and the separation solvent The same solvent as that used in the CS method was used to sample the structural isomer mixture of volatile compounds. The separation of the eluates was carried out.

[0091] (1) Method As a structural isomer mixture sample, (+)-isomene, a highly volatile monoterpene, was used. M A solution containing 10 mg / mL of each compound was prepared in TBE and subjected to SFC under the conditions shown in Table 12. As in Example 3, the SFC device was a Shimadzu Nexera UC (Shimadzu Corporation). The separated compounds were collected using a fraction collector FRC-40 SF (Shimadzu Corporation). To improve the sample recovery efficiency, a make-up filter was added to the fraction collector. The upsolvent was added at a constant flow rate.

[0092] [Table 12]

[0093] (2) Results The chromatogram obtained under the above conditions is shown in Figure 36. A mixture of structural isomers (two volatile monoterpene compounds) was separated within 7 minutes. I was able to do it.

[0094] Example 15: Absolute stereostructure determination by CS method In Example 15, two structural isomers of the volatile monoterpene compounds separated and fractionated in Example 14 were The absolute three-dimensional structure was determined using the CS method.

[0095] (1) Method [(ZnCl2 )3(tpt)2·(n-hexane) a ] n 1 grain of crystalline sponge (size 1 00μm×100μm×100μm, density 1.3g / cm 3 The theoretical amount is: 1.3 μg) was transferred to a V-bottom 1.2 mL vial with n-hexane. The solution was removed, and 50 μL of methyl tert-butyl ether (MTBE) was added to form crystals. The solvent in the vial was replaced with MTBE. It was set to the .

[0096] The components of the two peaks (peaks 1 and 2) shown in Figure 36 of Example 14 were determined from the above-mentioned crystal sponge. The mixture was directly dispensed into vials containing approximately 200 μg of each peak. After evaporating the solvent, the vial was capped, a syringe needle was inserted into the cap, and the vial was incubated at 50°C. The remaining solvent was slowly evaporated by stirring. After one day, the crystalline sponge was removed from the vial. The results were measured using a single crystal X-ray diffractometer (Cu Kα λ=1.5418 Å).

[0097] The measurement data was analyzed according to the method described in Chem. Eur. J. 2017, 23, 15035-15040.

[0098] (2) Results Measurements using a single crystal X-ray diffractometer and analysis of the measurement data revealed that the The structure of the guest compound was observed. Table 13 shows the crystal data obtained by analyzing the crystalline sponge containing the compound eluted by the method. show.

[0099] [Table 13]

[0100] When analyzing crystalline sponge incorporating compounds eluted as peaks 1 and 2, The crystal structures of the asymmetric units are shown in Figures 37 and 38, respectively. The molecules enclosed by dotted lines are the crystal structures. The guest structures observed are shown in Figure 39. and Figure 40. From these results, it was found that peak 1 was (+)-isomenthone ((2R, 5R)-5-methyl-2-(propan-2-yl)cyclohexanone), peak 2 is (- )-Isopulegol ((1R,2S,5R)-2-isopropenyl-5-methylcyclohexane From the results of Examples 14 and 15, it was possible to determine and identify the crystal structure of the compound. The volatile compounds separated by SFC were collected directly into a vial containing a syringe and then transferred to a crystalline sponge. It was confirmed that it is possible to incorporate the material and perform structural analysis.

[0101] Example 16: Separation of Volatile Structural Isomer Mixture Samples by SFC (2) In Example 16, the same SFC equipment and fraction collector as in Example 3 were used, and the separation solvent The same solvent as that used in the CS method was used to sample the structural isomer mixture of volatile compounds. The separation of the eluates was carried out. (1) Method As a structural isomer mixture sample, the volatile sesquiterpene (+)-β-eudesulfuric acid (Fujifilm Wako Pure Chemical Industries, Ltd.) and (-)-α-bisabolol (Sigma-Aldrich Co., Ltd.) ) to prepare a solution containing 10 mg / mL of each compound in MTBE, and test under the conditions in Table 14. As in Example 3, the SFC apparatus was a Shimadzu Nexera UC ( The separated compounds were collected using a fraction collector FRC-40 SF (Shimadzu Corporation). The fraction was collected by a fraction collection system (Shimadzu Corporation). Make-up solvent was added to the kuta at a constant flow rate.

[0102] [Table 14]

[0103] (2) Results The chromatogram obtained under the above conditions is shown in Figure 41. The structural isomer mixture sample (two volatile sesquiterpene compounds) was separated within 9 minutes. I was able to do this.

[0104] Example 17: Absolute stereostructure determination by CS method In Example 17, two structural isomers of the volatile sesquiterpene compounds separated and fractionated in Example 16 were The absolute three-dimensional structure of the compound was determined by the CS method.

[0105] (1) Method [(ZnCl2 )3(tpt)2·(n-hexane) a ] n 1 grain of crystalline sponge (size 1 00μm×100μm×100μm, density 1.3g / cm 3 The theoretical amount is: 1.3 μg) was transferred to a V-bottom 1.2 mL vial with n-hexane. The solution was removed, and 50 μL of methyl tert-butyl ether (MTBE) was added to form crystals. The solvent in the vial was replaced with MTBE. It was set to the .

[0106] The components of the two peaks (peaks 1 and 2) shown in Figure 41 of Example 16 were determined from the above-mentioned crystal sponge. The mixture was directly dispensed into vials containing approximately 200 μg of each peak. After evaporating the solvent, the vial was capped, a syringe needle was inserted into the cap, and the vial was incubated at 50°C. The remaining solvent was slowly evaporated by stirring. After one day, the crystalline sponge was removed from the vial. The results were measured using a single crystal X-ray diffractometer (Cu Kα λ=1.5418 Å).

[0107] The measurement data was analyzed according to the method described in Chem. Eur. J. 2017, 23, 15035-15040.

[0108] (2) Results Measurements using a single crystal X-ray diffractometer and analysis of the measurement data revealed that the The structure of the guest compound was observed. Table 15 shows the crystal data obtained by analyzing the crystalline sponge containing the compound eluted by the method. show.

[0109] [Table 15]

[0110] When analyzing crystalline sponge incorporating compounds eluted as peaks 1 and 2, The crystal structures of the asymmetric units are shown in Figures 42 and 43, respectively. The molecules enclosed by dotted lines are the crystal structures. The guest structures observed are shown in Figure 44. and Figure 45. From these results, it was found that peak 1 was (+)-β-eudesmol (( 3R,4aS)-Decahydro-5-methylene-α,α,8aβ-trimethyl-3β-naphtho Peak 2 is (-)-α-bisabolol ((2S)-6-methyl-2 -[(1S)-4-Methylcyclohex-3-en-1-yl]hept-5-ene-2- From the results of Examples 16 and 17, it was possible to determine and identify the crystalline sponge. Volatile compounds separated by SFC were collected directly into a vial containing crystalline sponge. It was confirmed that structural analysis was possible.

[0111] Example 18: Preparation of isopropanol-resistant crystalline sponge When isopropanol is used as the separation solvent for SFC, [(ZnCl2)3( tpt)2·(n-hexane) a ] n Type crystalline sponge is isopropanol resistant Therefore, in Example 18, the SFC and CS methods were not performed seamlessly. We investigated the preparation of a crystalline sponge that is resistant to isopropanol.

[0112] (1) Method 4,4'-bipyridine according to the method described in J. Am. Chem. Soc. 2015, 137, 12045-12049 (S)-mandelic acid (S-man) or (R)-mandelic acid (Rm an) and [Co2(R-man)2(bpy)3](N O3)2-type crystalline sponge and [Co2(S-man)2(bpy)3](NO3 Two types of crystalline sponges were prepared. These crystalline sponges were then soaked in isopropanol for several days. After immersion in water, the crystal structure was analyzed.

[0113] (2) Results [Co2(R-man)2(bpy)3](NO3)2 type crystalline sponge and [ The crystalline sponge of the Co2(S-man)2(bpy)3](NO3)2 type is isopropyl The crystal structure can be measured and observed with high accuracy even after immersion in alcohol for several days. It was found that (Figure 46).

[0114] Example 19: Enantiomeric separation of volatile racemates by SFC In Example 19, the same SFC equipment and fraction collector as in Example 3 were used, and the separation solvent In this study, the same solvent as that used in the CS method was used to separate the enantiomers of the volatile racemic compound. Ta.

[0115] (1) Method As a volatile racemic sample, rac-terpine, a volatile monoterpene racemic 4-Hydroxybenzone (Fujifilm Wako Pure Chemical Industries, Ltd.) was used, and the solution was diluted to 20 mg / mL with isopropanol. The sample was prepared and subjected to SFC under the conditions shown in Table 16. As in Example 3, the SFC apparatus was a Shimadzu The separated compounds were collected using a Nexera UC (Shimadzu Corporation). The samples were collected using a FRC-40 SF (Shimadzu Corporation). To achieve this, a constant flow of make-up solvent was added to the fraction collector.

[0116] [Table 16]

[0117] (2) Results The chromatogram obtained under the above conditions is shown in Figure 47. The two enantiomers of the terpene racemic compound could be separated within 5 min.

[0118] Example 20: Absolute stereostructure determination by CS method In Example 20, the enantiomers separated and isolated in Example 19 were compared with the results prepared in Example 18. The absolute stereostructure was determined by the CS method using a crystalline sponge.

[0119] (1) Method [Co2(S-man)2(bpy)3](NO3)2 type crystals prepared in Example 18 One sponge (size 100 μm × 100 μm × 100 μm, density 1.4 g / cm m 3 The theoretical amount when calculated is 1.4 μg, stored in chloroform until use) into a V-bottom 1. The mixture was transferred to a 2 mL vial together with chloroform. The chloroform was removed and isopropanol was added. 50 μL of isopropanol was added to the vial, and the solvent in the crystalline sponge was replaced with isopropanol. Several filters were prepared and placed in a fraction collector.

[0120] The components of the two peaks (peaks 1 and 2) shown in Figure 47 of Example 19 were determined from the above-mentioned crystal sponge. The mixture was dissolved gently under a nitrogen stream and directly dispensed into vials containing 100 µg of PEG (approximately 200 µg of each peak). After evaporating the solvent to about 50 μL, the vial was capped and incubated at 50°C for 3 days. After 3 days, the crystalline sponge was removed from the vial and analyzed by a single crystal X-ray diffractometer (Cu Kα λ = 1.5418 Å).

[0121] The measurement data are published in Chem. Eur. J. 2017, 23, 15035-15040 and J. Am. Chem. Soc. 2015, 1 37, 12045-12049.

[0122] (2) Results Measurements using a single crystal X-ray diffractometer and analysis of the measurement data revealed that the The structure of the guest compound was observed. Table 17 shows the crystal data obtained by analyzing a crystalline sponge containing compounds eluted by the method described above. show.

[0123] [Table 17]

[0124] When analyzing crystalline sponge incorporating compounds eluted as peaks 1 and 2, The crystal structures of the asymmetric units are shown in Figures 48 and 49, respectively. The molecules enclosed by dotted lines are the crystal structures. The guest structures observed are shown in Figure 50. and Figure 51. From these results, it was found that the enantiomer of peak 1 was S-terpinene-4 -ol ((4S)-4-isopropyl-1-methyl-1-cyclohexen-4-ol ), and the enantiomer of peak 2 is R-terpinen-4-ol ((4R)-4-isopropyl It was determined and identified as 1-methyl-1-cyclohexen-4-ol. From the results of Examples 8, 19 and 20, it was found that the use of isopropanol-resistant crystalline sponge The sample was separated by SFC (with isopropanol in the mobile phase) in a vial containing crystalline sponge. It is possible to directly recover the volatile compounds that have been released, incorporate them into the crystalline sponge, and analyze their structure. It was confirmed that this is the case.

[0125] Example 21: When isopropanol was removed by nitrogen gas flow, the results were compared using a conventional crystalline sponge. Absolute stereostructure determination by the CS method Isopropanol is [(ZnCl2)3(tpt)2] n Destroys type crystal sponge Therefore, in Example 21, isopropanol contained in the fractionated solution was removed by nitrogen gas. Is it possible to determine the absolute stereostructure of volatile compounds by the CS method when a process for removing volatile compounds is included? Specifically, the following steps were taken:

[0126] First, place an empty vial in the fraction collector and separate the two vials shown in Figure 47 of Example 19. Each component of the peaks (peaks 1 and 2) was collected there (approximately 200 μg of each peak). After removing the isopropanol contained in the solution by nitrogen gas flow (isopropanol is [(Zn Cl2)3(tpt)2] n destroys the crystalline sponge type), [(ZnCl2 )3(tpt)2] n A crystalline sponge of this type was added along with 50 μL of MTBE. Put a lid on the container, insert a syringe needle into the lid, and incubate at 50°C to gently remove the remaining solvent. After one day, the crystalline sponge was removed from the vial and analyzed by a single crystal X-ray diffractometer (C u Kα λ=1.5418Å), but the target compound could not be observed. The reason for this is that the volatile analyte was removed from the fraction in the process of removing isopropanol. It was thought that this was because the compounds were also lost.

[0127] Therefore, as shown by the results of Examples 18, 19, and 20, the SFC and CS methods can be seamlessly combined. It has been confirmed that performing the analysis in a single step is particularly advantageous when analyzing trace volatile compounds. Ta.

Claims

1. A method for determining the structure of a target substance contained in a mixture of two or more substances, comprising: (A) a step of separating the target substance from the mixture by supercritical fluid chromatography; (B) a step of adding a solvent for immersing the target substance separated in step (A) in a crystalline sponge to a solvent of a mobile phase of the supercritical fluid chromatography containing the target substance separated in step (A), and then immersing the separated target substance in the crystalline sponge to prepare a sample for crystal structure analysis; and (C) a step of performing crystal structure analysis of the sample for crystal structure analysis, wherein the solvent of the mobile phase of the supercritical fluid chromatography is a volatile solvent that does not contain water, and the crystalline sponge is a volatile solvent containing [(ZnX ２ ) ３ (tpt) ２ ・(solvent) ａ ] ｎ In the case of the [CuBr(btt)] type (wherein X represents a halogen atom such as chlorine, bromine, iodine, or fluorine, tpt represents 2,4,6-tri(4-pyridyl)-1,3,5-triazine, solvent represents a solvent contained in the pores, a represents any number equal to or greater than 0, and n represents any positive integer), the solvent of the mobile phase of the supercritical fluid chromatography is an ether, and the crystalline sponge is a [CuBr(btt)] type (wherein btt represents benzene-1,3,5-triyltriisonicotinic acid), [Co ２ (R-man) ２ (bpy) ３ ](NO ３ ) ２ type (where R-man represents (R)-mandelic acid and bpy represents 4,4'-bipyridine) or [Co ２ (S-man) ２ (bpy) ３ ](NO ３ ) ２ type (wherein S-man represents (S)-mandelic acid, and bpy represents 4,4'-bipyridine), the solvent of the mobile phase of the supercritical fluid chromatography is a nitrile and / or an alcohol.

2. The method of claim 1 , wherein the substance of interest is a volatile compound.

3. The octanol / water partition coefficient (log P ｏｗ 3. The method according to claim 1, wherein the value of (a) is -4.6 or more.

4. The method according to any one of claims 1 to 3, wherein the substance of interest is an enantiomer.

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