Separation method of cis-trans isomerized norrosewood

Abstract

The invention relates to the technical field of chemical separation, and discloses a separation method of cis-trans isomerized norrosewood, cis-trans norrosewood isomers are separated in high-speed counter-current chromatography through terpene-terpene DES, and a system with a separation effect on cis-trans isomerized norrosewood is designed through experiments. The method provided by the invention proves the feasibility of terpene-terpene DES on cis-trans isomer separation in high-speed counter-current chromatography, provides more choices for development of a new green solvent system, makes up for the vacancy of hydrophilic DES on separation of water-insoluble target objects in high-speed counter-current chromatography application, and can overcome the defects that a traditional organic solvent is large in use amount in the separation process, the separation time is short and the like. The residue is easy, and the environmental pollution is caused.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical separation, and specifically to a method for separating cis-trans isomeric norbixin. Background Art

[0002] Bixa orellana L., also known as annatto tree, is a deciduous small tree or shrub native to tropical America and is now widely distributed in tropical and subtropical regions of the world. It is a well-known tropical dyeing plant and is distributed in Yunnan, Fujian, Guangzhou, Guangxi and other places in China. Annatto seeds usually consist of 1.0 - 6.3% pigment, 9.6 - 13.3% moisture, 5.4 - 6.9% ash, 2.0 - 4.8% lipid, 12.1 - 17.0% protein and 70.0% total carbohydrates. The annatto pigment extracted from annatto seeds is divided into water-soluble and oil-soluble types according to different extraction methods. The main pigments are bixin and norbixin, which exist naturally in the form of cis-isomers and can be converted into trans-forms under the influence of heat and light.

[0003] Terpene-terpenoid deep eutectic solvents (DES) generally refer to stable solvents formed by the intermolecular hydrogen bonding association and melting of two or more substances, which are generally liquids at room temperature. One of them serves as a hydrogen bond acceptor (HBA), and the other serves as a hydrogen bond donor (HBD). DES has the advantages of simple preparation, low price, environmental friendliness, easy adjustment, good biocompatibility, good chemical stability, and recyclability, which fully meet the requirements of green chemistry and are widely used as green solvents in the field of organic synthesis, but there are few literature reports in the field of analytical chemistry.

[0004] Counter-current chromatography (CCC) is a liquid-liquid partition chromatography technique that does not require a solid support. Compared with traditional liquid-solid separation techniques, it eliminates some side effects caused by the solid support matrix and is relatively economical and environmentally friendly. High-speed counter-current chromatography (HSCCC) is based on CCC and has been greatly improved in terms of separation efficiency, separation time and sample loading capacity. It can achieve separation from the microgram level to the gram level, can effectively separate a large number of samples in a short time, and is commonly used for the separation and purification of natural product crude extracts and the intermediate separation of crude samples. It can also be combined with infrared spectroscopy and mass spectrometers, and has broad application prospects.

[0005] The HSCCC technology has become increasingly mature and perfect nowadays and is widely used in the separation and preparation of active ingredients in natural products. The extracts separated by it mainly include terpenoids, flavonoids, alkaloids, lignans, coumarins, saponins, polyphenols and anthraquinone derivatives. However, most of the systems used in high-speed counter-current chromatography technology are traditional organic solvents, which are used in large amounts, are prone to residues and can easily cause environmental pollution. The application of terpene-terpenoid deep eutectic solvents as green solvents in counter-current chromatography fully meets the requirements of green chemistry.

[0006] It is understood that there are currently no domestically produced bixin standards on the domestic market. A small number of standards are imported products from abroad, which are expensive. Moreover, there is currently no research report on the separation of norbixin isomers by the HSCCC method based on DES. Summary of the Invention

[0007] (1) Technical problems to be solved

[0008] In view of the deficiencies of the prior art, the present invention provides a method for separating cis-trans norbixin. The cis-trans norbixin isomers are separated by terpene-terpenoid DES in high-speed counter-current chromatography, thus solving the problem of separating cis-trans norbixin isomers.

[0009] (2) Technical solutions

[0010] In order to achieve the above object, the present invention discloses a method for separating cis-trans norbixin, which includes the following steps:

[0011] (1) Preparation of the sample solution: Dissolve the cis-trans norbixin sample in the mobile phase to obtain the sample solution;

[0012] (2) Selection of the solvent system: Take the solvent system in a centrifuge tube, shake well, let it stand for layering, add the sample solution to the solvent system, shake well, centrifuge for layering, and perform HPLC analysis to measure the distribution coefficient K of cis-trans norbixin in each system;

[0013] (3) Separation process of high-speed counter-current chromatography: First, pump the stationary phase into the column at a flow rate of 2 mL / min. After the high-speed counter-current chromatography column is filled with the stationary phase, rotate it, and then pump the mobile phase at a flow rate of 0.5 mL / min. After achieving dynamic equilibrium between the stationary phase and the mobile phase, inject 0.5 mL of the sample solution into the injection loop, monitor it in real time with a UV detector, and collect the fractions with an automatic fraction collector.

[0014] Preferably, the concentration of the sample prepared in step (1) is 1 mg / mL.

[0015] Preferably, the specific steps in step (2) are as follows: Take 2 mL of the prepared solvent system in a centrifuge tube, shake well and mix, let it stand for layering, add 10 μL of the sample solution to the system, shake well by oscillation, centrifuge for layering, take 200 μL each from the upper and lower phases for HPLC analysis, and determine the distribution coefficient K of cis- and trans-bixin in each system;

[0016] The distribution coefficient is calculated according to formula Ⅰ:

[0017] K = A U / A L Formula Ⅰ,

[0018] where A U is the peak area of the sample contained in the upper phase, and A L is the peak area of the sample contained in the lower phase.

[0019] Preferably, in step (2), during the preparation process of the solvent system, a suitable solvent system is selected according to the distribution coefficient, prepared in a separatory funnel according to the volume ratio composition in the solvent system, allowed to stand for 1 h for layering after being shaken well, and after separating the upper and lower phases before use, the two phases are ultrasonically degassed for 30 min respectively and waiting for use.

[0020] Preferably, the solvent system in step (2) consists of n-heptane, camphor + terpineol, and ammonia water, where the volume ratio of n-heptane, camphor + terpineol, and ammonia water is 2:3:5.

[0021] Preferably, the concentration of ammonia water in the solvent system in step (2) is 1.8 - 3.6 μmol / L.

[0022] Preferably, in step (2), under this solvent system, the stationary phases with flow rates of 0.5 mL / min, 1 mL / min, and 2 mL / min have retention rates of 51.1%, 60%, and 66.6% respectively.

[0023] Preferably, in step (3), during the separation process of high-speed countercurrent chromatography, the column volume is 27 mL, the host rotation speed is 1600 r / min, the upper phase is the stationary phase, the lower phase is the mobile phase, the flow rate of the pump for the stationary phase is 2 mL / min, and the flow rate of the pump for the mobile phase is 0.5 mL / min; the sample amount is 0.5 mg.

[0024] Preferably, in step (3), during the real-time monitoring process with a UV detector, the wavelength of the UV detector is 487 nm, and the rate of collecting fractions by the automatic fraction collector is 1 min / tube.

[0025] Preferably, in step (3), at equilibrium, the retention rate of the stationary phase is R f , R f is calculated by formula Ⅱ:

[0026] R f = V S / V C Formula II

[0027] where V S is the volume of the stationary phase in the high-speed counter-current chromatography column, and V C is the volume of the column of the high-speed counter-current chromatography column.

[0028] Preferably, in the step (3), the eluted fraction is detected and analyzed by HPLC;

[0029] The HPLC condition analysis is as follows:

[0030] Chromatographic column: Waters Symmetry C18 Column, specification 3.9 mm × 150 mm, 5 μm;

[0031] Mobile phase: Mobile phase A is 1% formic acid in water, and mobile phase B is acetonitrile; the proportions of the mobile phases are all in volume percentages. Gradient elution is adopted, and the mobile phase is composed of 80% mobile phase B and 20% mobile phase A, and the isocratic elution time is 6 min;

[0032] Flow rate: 0.5 mL / min;

[0033] Column temperature: 30 °C;

[0034] Injection volume: 10 μL;

[0035] Detection wavelength: 487 nm.

[0036] (III) Beneficial technical effects

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] (1) In the present invention, a system with a separation effect on cis-trans isomerized bixin is designed through experiments, which proves the feasibility of the separation of cis-trans isomers by terpene-terpene DES in high-speed counter-current chromatography. The application of terpene-terpene DES to high-speed counter-current chromatography provides more choices for the development of new green solvent systems, and at the same time makes up for the gap in the separation of water-insoluble targets by hydrophilic DES in HSCCC applications. It can overcome the disadvantages of traditional organic solvents, such as large usage amount, easy residue, and environmental pollution during the separation process. The separation of cis-trans isomers by terpene-terpene DES in high-speed counter-current chromatography proves the feasibility of the separation of cis-trans isomers by terpene-terpene DES in high-speed counter-current chromatography.

[0039] (2) The use of high-speed countercurrent chromatography in the present invention without using solids has many advantages. It can reduce the interaction between solute molecules and solid supports to retain the stationary phase, avoid irreversible adsorption, denaturation and inactivation of samples, and has a high recovery rate. The samples in the column can be completely recovered by various elution methods. Further, the requirement for sample pretreatment is low, and low-content crude samples can be directly loaded without causing any damage to the column. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0041] Figure 1 It is the elution curve diagram in Example 2 of the present invention;

[0042] Figure 2 It is the elution curve diagram in Example 3 of the present invention;

[0043] Figure 3 It is the elution curve diagram in Example 4 of the present invention;

[0044] Figure 4 It is the elution curve diagram in Example 5 of the present invention;

[0045] Figure 5 It is the elution curve diagram in Example 6 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0046] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in combination with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0047] Example 1

[0048] A method for separating cis-trans nor-bixin includes the following steps:

[0049] (1) Preparation of the sample solution: Take the cis-trans nor-bixin sample and dissolve it in the mobile phase, and the mobile phase is ammonia water with different concentrations to obtain the sample solution;

[0050] (2) Solvent system selection: Place the solvent system in a centrifuge tube. The solvent system consists of n - heptane, camphor + terpineol, and ammonia water with a volume ratio of 2:3:5. The molar ratio of camphor to terpineol is 1.5:1. Shake well to mix, let it stand for layering. Add the sample solution to the solvent system, shake well, centrifuge for layering, and perform HPLC analysis to determine the distribution coefficient K of cis - and trans - norbixin in the solvent system.

[0051] Perform high - speed counter - current chromatography separation on cis - and trans - isomeric norbixin. The separation conditions are as follows: the column volume is 27 mL; the main engine rotation speed is 1600 r / min; the upper phase is the stationary phase, and the lower phase is the mobile phase. The flow rate of the pump for the stationary phase is 2 mL / min, the flow rate of the pump for the mobile phase is 0.5 mL / min, the sample amount is 0.5 mg, use a UV detector for real - time monitoring, the wavelength of the UV detector is 487 nm, the rate of the automatic fraction collector for collecting fractions is 1 min / tube, and the retention rate R of the stationary phase f = 60%. Analyze the fractions by HPLC and obtain the elution curve.

[0052] Example 2

[0053] Based on the separation method of cis - and trans - isomeric norbixin in Example 1, in this system, the mobile phase is 1.8 μmol / L ammonia water. During the elution process, without recycling, the elution curve is as Figure 1 shown.

[0054] Example 3

[0055] Based on the separation method of cis - and trans - isomeric norbixin in Example 1, in this system, the mobile phase is 1.8 μmol / L ammonia water. During the elution process, recycle 3 times, and the elution curve is as Figure 2 shown.

[0056] Example 4

[0057] Based on the separation method of cis - and trans - isomeric norbixin in Example 1, in this system, the mobile phase is 1.8 μmol / L ammonia water. During the elution process, recycle 5 times, and the elution curve is as Figure 3 shown.

[0058] Example 5

[0059] Based on the separation method of cis - and trans - isomeric norbixin in Example 1, in this system, the mobile phase is 3.6 μmol / L ammonia water. During the elution process, recycle 5 times, and the elution curve is as Figure 4 shown.

[0060] Example 6

[0061] Based on the separation method of cis-trans isomer norbixin in Example 1, in this system, the mobile phase is 2.7 μmol / L ammonia water. During the elution process, it is circulated 5 times, and the elution curve is obtained as Figure 5 shown.

[0062] In the examples of the present invention, the sample solution is obtained by dissolving the cis-trans isomer norbixin sample in the mobile phase and mixing them, where the sample concentration is 1 mg / mL.

[0063] As Figures 1-3 shown, in this system, when the ammonia water concentration is 1.8 μmol / mL, the proportion of the overlapping area in the elution curve to the trans-norbixin first increases and then decreases with the number of cycles. When the number of cycles is large, the difference in the proportion is not significant. As Figures 3-5 shown, keeping the number of cycles at 5 times, when the ammonia water concentration increases, the proportion of the overlapping area in the elution curve to the trans-norbixin first increases and then decreases. Therefore, the ammonia water concentration of 2.7 μmol / mL is the most suitable.

[0064] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. All equivalent changes and improvements made within the scope of the application of the present invention shall still fall within the scope covered by the present invention.

Claims

1. A method for separating cis- and trans-isomers of norbixin, characterized in that: The steps include: (1) Preparation of sample solution: dissolve the cis- and trans-isomers of norbixin in the mobile phase to obtain a sample solution; (2) Selection of solvent system: put the solvent system into a centrifuge tube, shake and mix, let stand to separate, add the sample solution into the solvent system, shake and mix, centrifuge and separate, perform HPLC analysis, and determine the distribution coefficient K of cis-trans norbixin in different systems; (3) Separation process of high-speed countercurrent chromatography: First, the stationary phase is pumped into the column at a flow rate of 2 mL / min. When the high-speed countercurrent chromatography column is filled with the stationary phase, it is rotated, and then the mobile phase is pumped in at a flow rate of 0.5 mL / min. After dynamic equilibrium is achieved between the stationary phase and the mobile phase, 0.5 mL of sample solution is injected into the injection loop, monitored in real time by a UV detector, and the fractions are collected by an automatic fraction collector.

2. The method for separating cis- and trans-isomers of norbixin according to claim 1, characterized in that: In the step (1), the mobile phase is any one of 1.8 μmol / mL ammonia water, 2.7 μmol / mL ammonia water, and 3.6 μmol / mL ammonia water, and the prepared sample concentration is 1 mg / mL.

3. The method for separating cis- and trans-isomers of norbixin according to claim 1, characterized in that: The specific steps in step (2) are as follows: take 2 mL of the prepared solvent system into a centrifuge tube, shake and mix, let stand to separate layers, add 10 μL of the sample solution into the system, shake and mix, centrifuge and separate layers, take 200 μL of the upper and lower phases for HPLC analysis, and determine the distribution coefficient K of cis-trans norbixin in each system; The distribution coefficient is calculated according to formula Ⅰ: K=A U / A L Formula I, Among them A U is the peak area of ​​the sample contained in the upper phase, A L is the peak area of ​​the sample contained in the lower phase.

4. The method for separating cis- and trans-isomers of norbixin according to claim 1, characterized in that: In the step (2), during the preparation of the solvent system, different systems include an n-heptane system, a camphor+pinene alcohol system, and an ammonia system, wherein different ammonia concentrations result in different distribution coefficients K values. The solvent system is prepared in a separatory funnel according to the volume ratio of the components in the solvent system, and after being fully shaken, it is allowed to stand for 1 hour to separate the layers. Before use, the upper and lower phases are separated, and the two phases are degassed by ultrasonication for 30 minutes respectively before use.

5. The method for separating cis- and trans-isomers of norbixin according to claim 1, characterized in that: In the step (3), at equilibrium, the retention rate of the stationary phase is R f , R f Calculated by formula II: R f =V S / V C Formula II, Where V S is the volume of the stationary phase in the high-speed countercurrent column, V C is the volume of the high-speed countercurrent chromatography column.

6. The method for separating cis- and trans-isomers of norbixin according to claim 1, characterized in that: In the step (3), the effluent fractions of cis- and trans-norbixin are detected and analyzed by HPLC; The HPLC analysis conditions are: Chromatographic column: Waters Symmetry C18 Column, specification 3.9mm×150mm, 5μm; Mobile phase: Mobile phase A is 1% formic acid water, mobile phase B is acetonitrile; the mobile phase ratios are all volume percentages, gradient elution is adopted, the mobile phase is composed of 80% mobile phase B and 20% mobile phase A, and the isocratic elution time is 6 minutes; Flow rate: 0.5 mL / min; Column temperature: 30°C; Injection volume: 10 μL; Detection wavelength: 487nm.

7. The method for separating cis- and trans-isomers of norbixin according to claim 4, characterized in that: In the step (2), the solvent system consists of n-heptane, camphor+terpineol, and aqueous ammonia, wherein the volume ratio of n-heptane, camphor+terpineol, and aqueous ammonia is 2:3:5, and the molar ratio of camphor to terpineol is 1.5:

1.

8. The method for separating cis- and trans-isomers of norbixin according to claim 7, characterized in that: In the step (3), the flow rate of the mobile phase was adjusted to 0.5 mL / min, 1 mL / min, and 2 mL / min under this solvent system, and the retention rates of the stationary phase were 51.1%, 60%, and 66.6%, respectively.

9. The method for separating cis- and trans-isomers of norbixin according to claim 1, characterized in that: In the step (3), during the separation process of high-speed countercurrent chromatography, the column volume is 27 mL, the main engine speed is 1600 r / min, the upper phase is the stationary phase, the lower phase is the mobile phase, the pump stationary phase flow rate is 2 mL / min when the target is separated, and the pump mobile phase flow rate is 0.5 mL / min when the target is separated; the sample amount is 0.5 mg.

10. The method for separating cis- and trans-isomers of norbixin according to claim 1, characterized in that: In the step (3), during the real-time monitoring process using a UV detector, the wavelength of the UV detector is 487 nm, and the rate of collecting fractions by the automatic fraction collector is 1 min / tube.

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