Method for preparing menadione and heptaene menadione by using supercritical fluid chromatography

Abstract

The invention discloses a method for preparing menadione and heptaene menadione by utilizing supercritical fluid chromatography. The method for preparing the menadione and the heptaene menadione by using the supercritical fluid chromatography comprises the following steps: dissolving a sample containing the menadione and the heptaene menadione by using a dissolving reagent to prepare a test solution; carrying out supercritical fluid chromatographic separation on the test solution to respectively prepare the menadione and the heptaene menadione; the supercritical fluid chromatographic separation conditions are as follows: a mobile phase is a mixture of supercritical CO2 and an alkane solvent; the stationary phase comprises silica gel. The provided method for preparing menadione and heptaene menadione by using supercritical fluid chromatography realizes the separation of heptaene menadione and heptaene menadione, can reduce the use of organic reagents, and has the advantages of short separation time and good separation effect.

Description

Technical Field

[0001] The invention belongs to the field of chromatographic separation and purification, and relates to a method for preparing hexenylmenaquinone and heptenylmenaquinone by utilizing supercritical fluid chromatography. Background Art

[0002] Heptaquinone is a naturally occurring vitamin that contains the basic structure of menaquinone and has anti-hemorrhagic effects. It is a yellow crystal or oily liquid at room temperature. It is insoluble in water but soluble in organic solvents and vegetable oils. It is heat-resistant but easily destroyed by light. The structural formulas of hexenylmenaquinone and heptaquinone are as follows:

[0003] ,

[0004] .

[0005] Heptaquinone promotes the growth and proliferation of bone cells, strengthens bone growth and development, and maintains bone density. It also inhibits the activity of bone-resorbing cells, slowing bone loss and effectively preventing and improving osteoporosis. Heptaquinone can also activate substances such as Matrix Gla protein, preventing calcium ion deposition in arterial walls and regulating calcium ion concentrations in the blood, effectively reducing the risk of atherosclerosis. This function makes heptaquinone important in preventing cardiovascular disease. The traditional method for separating hexamethylenediamine and heptaquinone is high-performance liquid chromatography, which has drawbacks such as long separation times and the use of large amounts of chemical reagents. Summary of the Invention

[0006] Based on this, it is necessary to provide a method for preparing hexenylmenaquinone and heptaenylmenaquinone using supercritical fluid chromatography, which can achieve the separation of heptaenylmenaquinone, requires a shorter time, and reduces the amount of solvent used.

[0007] In some embodiments, a method for preparing hexamethylenequinone and heptaethylenequinone using supercritical fluid chromatography is provided, comprising the following steps:

[0008] Dissolve the sample containing hexamethylenequinone and heptaethylenequinone using a dissolving reagent to prepare a test solution;

[0009] The test solution is subjected to supercritical fluid chromatography to separate hexamethylene-methyl-menaquinone and heptaethylene-methyl-menaquinone respectively;

[0010] The conditions for supercritical fluid chromatography separation include: the mobile phase is a mixture of supercritical CO2 and an alkane solvent; and the stationary phase includes silica gel.

[0011] In some embodiments, the supercritical fluid chromatography separation conditions include: a back pressure of 10 MPa to 12 MPa.

[0012] In some embodiments, the alkane solvent includes one or more of n-hexane, heptane, pentane, cyclohexane, petroleum ether, and toluene.

[0013] In some embodiments, the supercritical fluid chromatography separation uses isocratic elution.

[0014] In some embodiments, during the isocratic elution process, the volume ratio of the supercritical CO2 to the alkane solvent is (90-95): (10-5).

[0015] In some embodiments, the supercritical fluid chromatography separation conditions include: a chromatographic column temperature of 25°C to 45°C.

[0016] In some embodiments, the supercritical fluid chromatography separation conditions include: a detection wavelength of 250 nm to 260 nm.

[0017] In some embodiments, the supercritical fluid chromatography separation conditions include: the flow rate of the mobile phase is 5 mL / min to 10 mL / min.

[0018] In some embodiments, the supercritical fluid chromatography separation conditions include: an injection volume of 1 μL to 200 μL.

[0019] In some embodiments, one or more of the following conditions are met:

[0020] (1) The dissolving agent includes one or more of n-hexane, heptane, pentane, cyclohexane, petroleum ether and toluene;

[0021] (2) The concentration of the test solution is 10 mg / mL to 100 mg / mL; and

[0022] (3) The chromatographic column is Nucifera SI, the stationary phase particle size is 5 μm, the chromatographic column diameter is 10 mm, and the chromatographic column length is 250 mm.

[0023] The aforementioned method for preparing hexamethylenediamine and heptaethylenediamine using supercritical fluid chromatography is based on the differences in the chemical properties of heptaethylenediamine and hexamethylenediamine. They have different distribution coefficients in a system consisting of a stationary phase and a mobile phase consisting of supercritical CO2 and an alkane solvent. When the two phases interact, these substances move with the mobile phase and are repeatedly distributed between the two phases, thereby achieving separation of the substances.

[0024] The provided method for preparing heptaenylmenadione using supercritical fluid chromatography achieves the separation of heptaenylmenadione and hexenylmenadione; CO2 is abundant and inexpensive, has low reactivity, low toxicity, and is non-flammable; the use of CO2 can reduce the use of organic reagents, lower costs, improve efficiency, be environmentally friendly, reduce harm to humans and the environment, and greatly reduce solvent treatment costs in subsequent drug production; the critical temperature and pressure of CO2 are compatible with chromatographs and are non-destructive to compounds to be analyzed and purified; the separation time is short, the separation effect is good, the purity of heptaenylmenadione can reach 99%, and the method has broad application prospects in the field of separation of heptaenylmenadione and hexenylmenadione. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments and examples of this application and to provide a more complete understanding of the application and its beneficial effects, the following briefly introduces the drawings required for use in the description of the embodiments or examples. Obviously, the drawings described below are only some embodiments of this application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0026] Figure 1 is the separation spectrum obtained in Example 1;

[0027] Figure 2 This is a chromatogram obtained by liquid chromatography in Example 1;

[0028] Figure 3 is the separation spectrum obtained in Comparative Example 1;

[0029] Figure 4 is the separation spectrum obtained in Comparative Example 2;

[0030] Figure 5 This is the separation spectrum obtained in Comparative Example 3. DETAILED DESCRIPTION

[0031] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the accompanying drawings. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0032] It should be noted that when an element is referred to as being “fixed to” another element, it may be directly on the other element or there may be an intermediate element. When an element is referred to as being “connected to” another element, it may be directly connected to the other element or there may be an intermediate element.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention pertains. The terms used in this specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0034] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0035] The terms "and / or", "or / and", and "and / or" used in this application include any one of two or more related listed items, and also include any and all combinations of the related listed items, and the said any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions: A, B and "a combination of A and B".

[0036] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0037] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0038] In this application, the "suitable" mentioned in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0039] In this application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute a limitation on the scope of protection of this application.

[0040] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0041] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0042] In the present invention, in the "first aspect," "second aspect," "third aspect," "fourth aspect," etc., the terms "first," "second," "third," "fourth," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, "first," "second," "third," "fourth," etc. serve only as non-exhaustive enumeration and description and should be understood not to constitute a closed-ended limitation on quantity.

[0043] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0044] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0045] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0046] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0047] The "room temperature" in this application generally refers to 5°C to 30°C, preferably 25±5°C.

[0048] Supercritical fluid chromatography (SFC) is a chromatographic method that uses a supercritical fluid as the mobile phase and a solid adsorbent as the stationary phase. Currently, SFC uses supercritical carbon dioxide in place of organic reagents. Leveraging the unique physicochemical properties of supercritical carbon dioxide, SFC has applications in stereoisomerization, petroleum product analysis, lipid and natural product separation, and pharmaceutical and food analysis.

[0049] The present invention discloses a method for preparing heptaenol using supercritical fluid chromatography. The method for preparing heptaenol using supercritical fluid chromatography comprises the following steps: dissolving a heptaenol sample using a dissolving reagent to prepare a test solution; subjecting the test solution to supercritical fluid chromatography separation to prepare heptaenol; the supercritical fluid chromatography separation conditions include: a mobile phase comprising a mixture of supercritical CO2 and an alkane solvent; and a stationary phase comprising silica gel. The method for preparing heptaenol using supercritical fluid chromatography achieves the separation of heptaenol and hexenol, can reduce the use of organic reagents, shortens the separation time, and achieves good separation effect.

[0050] In some embodiments, a method for preparing hexamethylenequinone and heptaethylenequinone using supercritical fluid chromatography is provided, comprising the following steps:

[0051] Dissolve the sample containing hexamethylenequinone and heptaethylenequinone using a dissolving reagent to prepare a test solution;

[0052] The test solution was subjected to supercritical fluid chromatography to prepare hexenylmenaquinone and heptaenylmenaquinone respectively;

[0053] The conditions for supercritical fluid chromatography separation include: the mobile phase is a mixture of supercritical CO2 and an alkane solvent; the stationary phase includes silica gel.

[0054] In some embodiments, the conditions for supercritical fluid chromatography separation include: a back pressure of 10 MPa to 12 MPa, for example, the back pressure can be 10 MPa, 11 MPa, 12 MPa, etc., or a range consisting of any two of the foregoing values.

[0055] In some embodiments, the alkane solvent includes one or more of n-hexane, heptane, pentane, cyclohexane, petroleum ether, and toluene.

[0056] In some embodiments, supercritical fluid chromatography separation employs isocratic elution.

[0057] In some embodiments, during the isocratic elution process, the volume ratio of supercritical CO2 to the alkane solvent is (90~95):(10~5), for example, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, etc., or a range consisting of any two of the foregoing ratios.

[0058] In some embodiments, the conditions for supercritical fluid chromatography separation include: a column temperature of 25°C to 45°C, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, etc., or a range consisting of any two of the foregoing values.

[0059] In some embodiments, the conditions for supercritical fluid chromatography separation include: a detection wavelength of 250 nm to 260 nm, for example, 250 nm, 251 nm, 252 nm, 253 nm, 254 nm, 255 nm, 256 nm, 257 nm, 258 nm, 259 nm, 260 nm, etc., or a range consisting of any two of the foregoing values.

[0060] In some embodiments, the conditions for supercritical fluid chromatography separation include: a flow rate of the mobile phase of 5 mL / min to 10 mL / min, for example, 5 mL / min, 6 mL / min, 7 mL / min, 8 mL / min, 9 mL / min, 10 mL / min, etc., or a range consisting of any two of the foregoing values.

[0061] In some embodiments, the conditions for supercritical fluid chromatography separation include: an injection volume of 1 μL to 200 μL, for example, 1 μL, 5 μL, 10 μL, 20 μL, 50 μL, 100 μL, 150 μL, 200 μL, etc., or a range consisting of any two of the foregoing values.

[0062] In some embodiments, the dissolving agent comprises one or more of n-hexane, heptane, pentane, cyclohexane, petroleum ether, and toluene.

[0063] In some embodiments, the concentration of the test solution is 10 mg / mL to 100 mg / mL, for example, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, etc., or a range consisting of any two of the foregoing values.

[0064] In some embodiments, the chromatographic column is Nucifera SI, the stationary phase particle size is 5 μm, the chromatographic column diameter is 10 mm, and the chromatographic column length is 250 mm.

[0065] In order to make it easier to understand and implement the present invention, the following easier-to-implement, more specific and detailed embodiments and comparative examples are provided as reference.

[0066] The following will further illustrate the concept, specific examples and technical effects of the present invention in conjunction with the accompanying drawings to fully understand the present invention. The purpose of providing these illustrations is only to help explain the present invention and should not be used to limit the scope of the claims of the present invention.

[0067] The following are specific examples, which describe the present disclosure in more detail. These examples are intended for illustrative purposes only, as various modifications and variations within the scope of the present disclosure will be apparent to those skilled in the art. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are by weight, and all reagents used in the examples are commercially available or synthesized according to conventional methods and can be used directly without further processing, and the instruments used in the examples are commercially available. Unless otherwise stated, the raw materials used in the following experiments are all commercially available.

[0068] Example 1

[0069] The chromatograph used in this example is a supercritical fluid chromatograph produced by Jiangsu Hanbang Technology Co., Ltd. The chromatographic column size is 10×250 mm, the mobile phase flow rate is 10 mL / min, the back pressure is set to 12 MPa, the concentration of the heptaquinone sample is 100 mg / mL, the injection volume is 200 μL, and the detection wavelength is 254 nm.

[0070] Table 1-1 shows other relevant conditions selected in the method for separating and preparing heptaquinone and hexenquinone using a supercritical fluid chromatography system in Example 1.

[0071] Table 1-1 SFC separation conditions in Example 1

[0072]

[0073] The specific steps corresponding to Example 1 are as follows: dissolving the heptaquinone sample in n-hexane, performing ultrasonic dissolution to prepare a 100 mg / mL solution, and filtering the solution through a nylon membrane with a pore size of 0.45 μm to obtain a test solution.

[0074] On a supercritical fluid chromatograph from Jiangsu Hanbang Technology Co., Ltd., Nucifera SI 5um 10×250mm (silica gel) was selected as the chromatographic column. The volume ratio of supercritical carbon dioxide and n-hexane in the mobile phase, total flow rate, back pressure, and column temperature were controlled according to Table 1-1. The temperature of carbon dioxide entering the pump was maintained at 6°C. The test solution was separated, and the separation effect was as follows. Figure 1 Table 1-2 is Figure 1 Retention time and peak area of ​​heptaquinone.

[0075] Table 1-2 Figure 1 Retention time and peak area of ​​heptaquinone

[0076]

[0077] like Figure 1 As shown in Table 1-2, the use of the above-mentioned supercritical fluid chromatography system to separate the test solution containing the heptadecene-methyl menadione sample can achieve baseline separation of heptadecene-methyl menadione and hexenyl-methyl menadione, wherein the peak with a retention time of 14.04 min is heptadecene-methyl menadione, and the peak with a retention time of 8.18 min is hexenyl-methyl menadione. The time required is shorter, which reduces the amount of solvent used.

[0078] The prepared heptaquinone was tested by external standard method using liquid chromatography. 100% methanol was used for testing on C18 packing, with a packing particle size of 5 μm, a chromatographic column diameter of 4.6 mm, and a chromatographic column length of 250 mm. A standard curve was drawn using the same method using the standard sample. The concentration of the prepared sample was substituted into the standard curve to obtain the peak area corresponding to the concentration. The peak area obtained by the prepared sample test was divided by the peak area obtained by substituting the concentration into the standard curve to obtain a detection purity of 99.1%. The test results are as follows: Figure 2 As shown in Table 1-3 Figure 2 Retention time and peak area of ​​heptaquinone.

[0079] Table 1-3 Figure 2 Retention time and peak area of ​​heptaquinone

[0080]

[0081] Comparative Example 1

[0082] The chromatograph used was a high-pressure liquid chromatograph produced by Jiangsu Hanbang Technology Co., Ltd. The column specifications were 10×250 mm, the mobile phase flow rate was 3 mL / min, the concentration of the heptaquinone sample was 40 mg / mL, the injection volume was 800 μL, and the detection wavelength was 254 nm.

[0083] Table 2-1 shows other relevant conditions selected in the method for separating and preparing heptaquinone and hexenquinone using a high pressure liquid chromatography system.

[0084] Table 2-1 HPLC separation conditions in Example 2

[0085]

[0086] The specific steps for separating and preparing heptaquinone are as follows: dissolve the heptaquinone sample in a solution of n-hexane, ethanol, and methanol in a volume ratio of 2:1:2, use ultrasonic dissolution to prepare a 40 mg / mL solution, and filter it with a nylon membrane with a pore size of 0.45 μm. On a high-pressure liquid chromatograph of Jiangsu Hanbang Technology Co., Ltd., Nucifera C18P10um 10×250mm (octadecyl bonded to the silica surface) is selected as the chromatographic column. According to Table 2-1, the volume ratio of methanol and water in the mobile phase, the total flow rate, and the column temperature are controlled to separate the heptaquinone sample. The separation effect is as follows: Figure 3 shown.

[0087] Table 2-2 Figure 3 Retention time and peak area of ​​heptaquinone

[0088]

[0089] like Figure 3 As shown in Table 2-2, the high pressure liquid chromatography system was used to separate and prepare heptaquinone, which could achieve baseline separation. Figure 3 The peak with a retention time of 83.617 min is heptaenylmenaquinone, and the peak with a retention time of 47.745 min is hexenylmenaquinone. The separation and preparation of heptaenylmenaquinone by this method requires a long time and a large amount of solvent.

[0090] Comparative Example 2

[0091] The chromatograph used in this comparative example is a supercritical fluid chromatograph produced by Jiangsu Hanbang Technology Co., Ltd. The chromatographic column specifications are 10×250 mm, the mobile phase flow rate is 10 mL / min, the back pressure is set to 12 Mpa, the concentration of the heptaquinone sample is 100 mg / mL, the injection volume is 200 μL, and the detection wavelength is 254 nm.

[0092] Table 3-1 shows other relevant conditions selected in the method for separating and preparing cis-heptenylmenaquinone and hexenylmenaquinone using a supercritical fluid chromatography system in Comparative Example 2.

[0093] Table 3-1 SFC separation conditions in Comparative Example 2

[0094]

[0095] The specific steps corresponding to Comparative Example 2 are as follows: dissolving the heptaquinone sample in n-hexane, performing ultrasonic dissolution to prepare a 100 mg / mL solution, and filtering with a nylon membrane with a pore size of 0.45 μm to obtain a test solution.

[0096] On the supercritical fluid chromatograph of Jiangsu Hanbang Technology Co., Ltd., Nucifera SI 5um 10×250mm (silica gel) was selected as the chromatographic column. The volume ratio of supercritical carbon dioxide and ethanol in the mobile phase, total flow rate, back pressure and column temperature were controlled according to Table 3-1. At the same time, the temperature of carbon dioxide entering the pump was ensured to be 6°C. The test solution was separated, and the separation effect was as follows: Figure 4 As shown, it can be clearly seen that the target objects cannot be separated.

[0097] Comparative Example 3

[0098] The chromatograph used in this comparative example is a supercritical fluid chromatograph produced by Jiangsu Hanbang Technology Co., Ltd. The chromatographic column specifications are 10×250 mm, the mobile phase flow rate is 10 mL / min, the back pressure is set to 12 Mpa, the concentration of the heptaquinone sample is 100 mg / mL, the injection volume is 200 μL, and the detection wavelength is 254 nm.

[0099] Table 4-1 shows other relevant conditions selected in the method for separating and preparing cis-heptenylmenaquinone and hexenylmenaquinone using a supercritical fluid chromatography system in Comparative Example 3.

[0100] Table 4-1 SFC separation conditions in Comparative Example 3

[0101]

[0102] The specific steps corresponding to Comparative Example 4 are as follows: dissolving the heptaquinone sample in n-hexane, ultrasonically dissolving it to prepare a 100 mg / mL solution, and filtering it with a nylon membrane with a pore size of 0.45 microns to obtain a test solution.

[0103] On the supercritical fluid chromatograph of Jiangsu Hanbang Technology Co., Ltd., Nucifera CN 5um 10×250mm (cyano group bonded to the silica gel surface) was selected as the chromatographic column. The volume ratio of supercritical carbon dioxide and n-hexane in the mobile phase, the total flow rate, the back pressure and the column temperature were controlled according to Table 4-1. At the same time, the temperature of carbon dioxide entering the pump was ensured to be 6°C. The test solution was separated. The separation effect was as follows: Figure 5 As shown, it can be seen that the separation effect is obviously not as good as that of Example 1.

[0104] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0105] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that a person of ordinary skill in the art may make various modifications and improvements without departing from the spirit of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims, and the description and drawings shall be used to interpret the content of the claims.

Claims

1. A method for preparing hexamethylenequinone and heptaethylenequinone using supercritical fluid chromatography, characterized in that: The following steps are involved: Dissolve the sample containing hexamethylenequinone and heptaethylenequinone using a dissolving reagent to prepare a test solution; The test solution is subjected to supercritical fluid chromatography to separate hexamethylene-methyl-menaquinone and heptaethylene-methyl-menaquinone respectively; The conditions for supercritical fluid chromatography separation include: the mobile phase is a mixture of supercritical CO2 and an alkane solvent; and the stationary phase includes silica gel.

2. The method according to claim 1, characterized in that The conditions for supercritical fluid chromatography separation include: a back pressure of 10 Mpa to 12 Mpa.

3. The method according to claim 1, characterized in that The alkane solvent includes one or more of n-hexane, heptane, pentane, cyclohexane, petroleum ether and toluene.

4. The method according to any one of claims 1 to 3, characterized in that The supercritical fluid chromatography separation adopts isocratic elution.

5. The method according to claim 4, characterized in that During the isocratic elution process, the volume ratio of the supercritical CO2 to the alkane solvent is (90~95): (10~5).

6. The method according to any one of claims 1 to 4, characterized in that The supercritical fluid chromatography separation conditions include: the chromatographic column temperature is 25°C to 45°C.

7. The method according to any one of claims 1 to 4, characterized in that The conditions for supercritical fluid chromatography separation include: a detection wavelength of 250 nm to 260 nm.

8. The method according to any one of claims 1 to 4, characterized in that The conditions for supercritical fluid chromatography separation include: the flow rate of the mobile phase is 5 mL / min to 10 mL / min.

9. The method according to any one of claims 1 to 4, characterized in that The conditions for supercritical fluid chromatography separation include: an injection volume of 1 μL to 200 μL.

10. The method according to any one of claims 1 to 4, characterized in that One or more of the following conditions are met: (1) The dissolving agent includes one or more of n-hexane, heptane, pentane, cyclohexane, petroleum ether and toluene; (2) The concentration of the test solution is 10 mg / mL to 100 mg / mL; and (3) The chromatographic column is Nucifera SI, the stationary phase particle size is 5 μm, the chromatographic column diameter is 10 mm, and the chromatographic column length is 250 mm.

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