Method for separating and purifying tocopherol monomer

Abstract

The invention discloses a method for separating and purifying tocopherol monomers. The method comprises the following steps: diluting natural vitamin E with cyclohexane, and carrying out chromatography treatment by using a first silica gel column, with an eluent comprising cyclohexane, ethanol and n-propanol in a ratio of 100: (0.1-0.3): (1-2); 14 equal parts of elution components are obtained in sequence; the elution components of the Fr3-Fr5, the Fr8-Fr9 and the Fr11-Fr12 are collected; carrying out vacuum concentration on the elution components of Fr8-Fr9 and Fr11-Fr12 to respectively obtain gamma-tocopherol and delta-tocopherol, and carrying out vacuum concentration on the elution component of Fr3-Fr5; carrying out chromatographic treatment by adopting a second silica gel column, directly loading a concentrated solution on the column, collecting an eluent which is cyclohexane-ethyl acetate in a ratio of 100: (70-85), and carrying out vacuum concentration on the eluent to obtain beta-tocopherol; and regenerating the second silica gel column by using cyclohexane, collecting an elution component, and concentrating under reduced pressure to obtain the alpha-tocopherol.

Description

Technical Field

[0001] This invention relates to the field of purification and separation technology of natural vitamin E, and specifically to a method for separating and purifying tocopherol monomers. Background Technology

[0002] Tocopherol is an essential fat-soluble vitamin for the human body, belonging to a large class of the vitamin E family. Based on the position and number of methyl groups on the chromoyl alcohol benzene ring, tocopherol can be classified into α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol. Naturally occurring tocopherols are generally mixtures of 3-4 monomers. β-tocopherol and γ-tocopherol are species-dependent and generally do not coexist in the same crop or oilseed.

[0003] In the prior art, β-tocopherol and γ-tocopherol are usually separated together.

[0004] For example, patent application number CN201110331857.8 discloses a method for separating and preparing high-purity natural vitamin E monomers from mixed tocopherols, including the following steps: 1) Adsorption: Dissolve 1 volume part of a mixed tocopherol with a mass concentration of 30-70% in 1-10 volume parts of mobile phase solvent at 20-60℃ to obtain a loading solution; pack a fixed reverse phase packing in a reverse phase chromatography column, and add the loading solution at 20-60℃ into the chromatography column to allow the loading solution to pass through the packing.

[0005] 2) Analysis: Add 1-10 times the volume of the sample liquid to the chromatography column as mobile phase solvent II; discard less than 10% of the eluent volume, collect more than or equal to 10% and less than 30% of the eluent volume as eluent I, remove the solvent to obtain eluent I with d-α-tocopherol purity ≥ 90%; collect more than or equal to 30% and less than 70% of the eluent volume as eluent II, remove the solvent to obtain eluent II with d-γ-tocopherol purity ≥ 90%; collect more than or equal to 70% of the eluent volume as eluent III, remove the solvent to obtain eluent III with d-δ-tocopherol purity ≥ 85%. The first effluent was subjected to molecular distillation to obtain d-α-tocopherol with a purity ≥95%; the second effluent was subjected to molecular distillation to obtain d-γ-tocopherol with a purity ≥95%; and the third effluent was subjected to molecular distillation to obtain d-δ-tocopherol with a purity ≥90%.

[0006] The patent has the following problems: First, it cannot separate (β+γ)-tocopherols; second, molecular distillation is required to obtain high-purity monomers; third, the three effluents are sequentially connected, the distinguishing point is small, and it is easy to miss the distinguishing point.

[0007] For example, patent application number CN201710395583.6 discloses a method for separating individual tocopherols from a mixture of tocopherols using a polyionic liquid. The adsorption separation method is either fixed-bed adsorption separation or simulated moving-bed adsorption separation. When the adsorption separation method is fixed-bed adsorption separation, it includes the following steps: (1) Pack the polyionic liquid into the chromatography column.

[0008] (2) Dissolve the mixed crude tocopherol product fully in elution solvent I to prepare the column loading solution. (3) The upper column solution is passed into the chromatography column at the set adsorption temperature. (4) At the set desorption temperature, eluent II was introduced into the chromatography column, the eluent was collected in segments, the eluents with the same components were combined, and the column was distilled under reduced pressure to obtain α-tocopherol, β-tocopherol and γ-tocopherol mixture, and δ-tocopherol, respectively.

[0009] (5) Continue to rinse the chromatography column with elution solvent I to regenerate the polyionic liquid and enter the next adsorption separation cycle.

[0010] The patent has the following problems: first, it requires the use of a special polyionic liquid; second, it cannot separate a mixture of β-tocopherol and γ-tocopherol.

[0011] For example, patent application number CN201810804465.0 discloses a method for separating and purifying high-purity tocopherol monomers from mixed tocopherols, including, Sample dissolution: The mixed tocopherols are ultrasonically dissolved into the mobile phase of the high-pressure preparative liquid phase system.

[0012] Sample loading: Load the sample into a well-equilibrated high-pressure preparative liquid phase system equipped with a UV detector. Sample collection: Based on the peak elution, the substance peaks are collected at regular intervals and in quantitative amounts to obtain a substance peak solution. Separation and purification of monomers: The peak solution of the substance was analyzed by an analytical high performance liquid chromatography system to obtain collection solutions with d-δ tocopherol purity ≥ 99%, d-(γ-+β)-tocopherol purity ≥ 99%, and d-α-tocopherol purity ≥ 98%, respectively. The mobile phase was removed from the obtained collection solutions to obtain the high-purity tocopherol monomers.

[0013] The patent has the following problems: first, it requires a high-pressure liquid phase preparation system; second, it cannot separate the mixture of β-tocopherol and γ-tocopherol. Summary of the Invention

[0014] This invention provides a method for separating and purifying tocopherol monomers, yielding four tocopherol monomers in a simple process. The patent employs a two-stage elution process. In the first elution, β-tocopherol and γ-tocopherol are separated using a specific eluent; however, this step cannot achieve complete separation of the four tocopherol monomers. It yields α+β-tocopherol, γ-tocopherol, and δ-tocopherol; however, α+β-tocopherol is particularly easy to separate. In the second elution, β-tocopherol can be obtained with simple elution. Because α+β-tocopherol contains very few impurities, α-tocopherol can be obtained during the regeneration process of the second elution. During the second elution and regeneration process, the overall yield of α+β-tocopherol is particularly high, and the purity of β-tocopherol is particularly high. The technical solution is as follows: This invention provides a method for the isolation and purification of tocopherol monomers, the method comprising the following steps: S101: Concentrate natural vitamin E to increase the tocopherol concentration to 40-65%. If the natural vitamin E itself has a high tocopherol concentration, concentration is not necessary. The natural vitamin E in this patent is a byproduct of processing soybean oil, corn oil, or rapeseed oil (e.g., produced during deodorization).

[0015] S102: Dilute the concentrate obtained in step S101 with cyclohexane and perform chromatography using a first silica gel column. The eluent is cyclohexane-ethanol-n-propanol (volume ratio) = 100:0.1-0.3:1-2. 14 equal eluent fractions (based on the eluent fraction elution time) are obtained and named Fr1-Fr14, respectively. Collect the eluent fractions Fr3-Fr5, Fr8-Fr9, and Fr11-Fr12. Concentrate the eluent fractions Fr8-Fr9 and Fr11-Fr12 under reduced pressure to obtain γ-tocopherol and δ-tocopherol, respectively. Concentrate the eluent fraction Fr3-Fr5 under reduced pressure until the tocopherol concentration reaches above 0.5 g / mL to obtain a first-stage chromatographic concentrate.

[0016] from Figure 1 It can be seen that impurities such as oils and methyl esters are eluted first; α+β-tocopherol, γ-tocopherol and δ-tocopherol are eluted in sequence and can be basically completely separated.

[0017] S103: Chromatography was performed using a second silica gel column. The concentrated solution from the first chromatography was directly loaded onto the column. The eluent was cyclohexane-ethyl acetate = 100:70-85. The eluent was collected (discarding the initial and final portions). The eluent was concentrated under reduced pressure to obtain β-tocopherol.

[0018] S104: The second silica gel column was regenerated with cyclohexane, the eluent was collected, and the α-tocopherol was obtained by vacuum concentration.

[0019] Both the first and second silica gel columns are low-pressure chromatography columns. Specifically, the first silica gel column has a height-to-diameter ratio of 15-25:1, and the silica gel used has a particle size of 100-200 mesh. The second silica gel column has a height-to-diameter ratio of 5-10:1, and the silica gel used has a particle size of 100-200 mesh.

[0020] In step S102, the concentrate is diluted with cyclohexane to a tocopherol concentration of 0.5-2.0 g / mL, the eluent flow rate is 3-8 column volumes / h, the sample loading amount is 80-150 mg / mL column volume, and one eluent fraction is collected every five minutes.

[0021] Furthermore, the first silica gel column is regenerated. The regeneration process is as follows: first, elution is performed with 1-3 column volumes of methanol, followed by elution with 1-3 column volumes of cyclohexane. Similar to conventional techniques, the number of regeneration cycles can generally reach more than 8.

[0022] In step S103, the flow rate of the eluent is 1-4 column volumes / h, and the sample loading volume is 70-120 mg / mL column volume.

[0023] In step S104, the second silica gel column is regenerated with 3-5 column volumes of cyclohexane to obtain 10 equal portions (based on the eluent component elution time), named FR1-FR10 respectively; the eluent components FR2-FR7 are collected and concentrated under reduced pressure to obtain α-tocopherol.

[0024] Preferably, in step S102, the eluent is cyclohexane-ethanol-n-propanol = 100:0.2:1; in step S103, the eluent is cyclohexane-ethyl acetate = 100:80.

[0025] The beneficial effects of the technical solution provided by the embodiments of the present invention are as follows: (1) α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol can be obtained respectively; (2) Steps S103 and S104 are simple elution processes, requiring only the elution of one component, with high yield and a total yield of more than 85%; (3) The purity of the obtained β-tocopherol is particularly high, reaching over 95% (tocopherol with a purity of over 95% is relatively expensive and can be used for laboratory analysis). (4) Under optimized conditions, γ-tocopherol had a purity of 92.4% and a yield of 41.7%; δ-tocopherol had a purity of 90.5% and a yield of 58.3%; β-tocopherol had a purity of 95.9% and a yield of 48.3%; and α-tocopherol had a purity of 88.3% and a yield of 45.4%. (5) Separating α-tocopherol during the regeneration process can reduce the number of steps. (6) The second silicone column can be regenerated at least 15 times. The second silicone column has a longer service life under simpler regeneration conditions, which can reduce costs and is suitable for industrial production. Attached Figure Description

[0026] Figure 1 This is the efflux curve of tocopherol; Figure 2 This is a line graph showing the CV values ​​of different eluent ratios on step S102. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0028] Example 1 Example 1 provides a method for the isolation and purification of tocopherol monomers, comprising the following steps: S101: Concentrate natural vitamin E (tocopherol content 37.1%) to increase the tocopherol concentration to 57.3%. The concentrated natural vitamin E has a tocopherol content of 57.3% (α-tocopherol 9.3%, β-tocopherol 5.1%, γ-tocopherol 26.3%, δ-16.6%), and is derived from deodorized soybean oil.

[0029] S102: Dilute the concentrated solution obtained in step S101 with cyclohexane to a tocopherol concentration of 1 g / mL, and perform chromatography using a first silica gel column. The first silica gel column is a low-pressure chromatography column with a column volume of 250 mL. The silica gel used has a particle size of 100-200 mesh. It is activated with cyclohexane and packed using a wet packing method. The injection volume is 3 mL, to the top of the silica gel column. The eluent flow rate is 20 mL / min. The eluent is pumped in and the timing is started. The eluent was cyclohexane-ethanol-n-propanol = 100:0.2:1; the elution time was 70 minutes; the eluent fraction eluted from 10 to 25 minutes was collected and concentrated under reduced pressure to a tocopherol concentration of 1.0 g / mL (parallel experiments were conducted simultaneously, and the eluent fractions from the parallel experiments were directly evaporated to dryness, and the yield and purity were calculated; the yield was 55.3% and the purity was 87.1%); the eluent fraction eluted from 35 to 45 minutes was collected and concentrated under reduced pressure to obtain γ-tocopherol with a purity of 92.4% and a yield of 41.7%; the eluent fraction eluted from 50 to 60 minutes was collected and concentrated under reduced pressure to obtain δ-tocopherol with a purity of 90.5% and a yield of 58.3%.

[0030] S103: Chromatography was performed using a second silica gel column. This second column was a low-pressure chromatography column with a volume of 200 mL. The silica gel used had a particle size of 100-200 mesh. Initial activation with cyclohexane was performed, and the column was packed using a wet packing method. The concentrate from the first chromatography step was directly loaded onto the column at an injection volume of 2 mL (combining concentrates from multiple parallel experiments in step S102). The eluent flow rate was 5 mL / min. The eluent was cyclohexane-ethyl acetate = 100:80; the elution time was 2.5 hours; the eluent was collected and concentrated under reduced pressure to obtain β-tocopherol with a purity of 95.9% and a yield of 48.3%.

[0031] S104: The second silica gel column was regenerated with 800 mL of cyclohexane for 3 hours; the eluent was collected after 18-126 minutes and concentrated under reduced pressure to obtain α-tocopherol with a purity of 88.3% and a yield of 45.4%.

[0032] In steps S102 to S103 and S104, the total yield of β-tocopherol and α-tocopherol was 87.8%.

[0033] Example 2 Example 2 provides a method for the isolation and purification of tocopherol monomers, comprising the following steps: S101: Concentrate natural vitamin E to increase the mass concentration of tocopherol (tocopherol content of 37.1%) to 57.3%. The concentrated natural vitamin E has a tocopherol content of 57.3% (α-tocopherol 9.3%, β-tocopherol 5.1%, γ-tocopherol 26.3%, δ-16.6%), and is derived from deodorized soybean oil.

[0034] S102: Dilute the concentrated solution obtained in step S101 with cyclohexane to a tocopherol concentration of 1 g / mL, and perform chromatography using a first silica gel column. The first silica gel column is a low-pressure chromatography column with a column volume of 250 mL. The silica gel used has a particle size of 100-200 mesh. Initially, it is activated with cyclohexane and packed using a wet packing method. The injection volume is 3 mL, to the top of the silica gel column. The eluent flow rate is 20 mL / min. Pump in the eluent and start timing. The eluent was cyclohexane-ethanol-n-propanol = 100:0.1:1.5; the elution time was 70 minutes; the eluent fraction eluted from 10 to 25 minutes was collected and concentrated under reduced pressure to a tocopherol concentration of 1.0 g / mL (parallel experiments were conducted simultaneously, and the eluent fractions from parallel experiments were directly evaporated to dryness, and the yield and purity were calculated; the yield was 31.4% and the purity was 95.5%); the eluent fraction eluted from 35 to 45 minutes was collected and concentrated under reduced pressure to obtain γ-tocopherol with a purity of 90.1% and a yield of 37.1%; the eluent fraction eluted from 50 to 60 minutes was collected and concentrated under reduced pressure to obtain δ-tocopherol with a purity of 87.4% and a yield of 60.3%.

[0035] S103: Chromatography was performed using a second silica gel column. This second column was a low-pressure chromatography column with a volume of 200 mL. The silica gel used had a particle size of 100-200 mesh. Initial activation with cyclohexane was performed, and the column was packed using a wet packing method. The concentrate from the first chromatography step was directly loaded onto the column at an injection volume of 2 mL (combining concentrates from multiple parallel experiments in step S102). The eluent flow rate was 5 mL / min. The eluent was cyclohexane-ethyl acetate = 100:70; the elution time was 2.5 hours; the eluent was collected and concentrated under reduced pressure to obtain β-tocopherol with a purity of 98.5% and a yield of 30.2%.

[0036] S104: The second silica gel column was regenerated with 800 mL of cyclohexane for 3 hours; the eluent was collected after 18-126 minutes and concentrated under reduced pressure to obtain α-tocopherol with a purity of 90.1% and a yield of 26.7%.

[0037] In steps S102 to S103 and S104, the total yield of β-tocopherol and α-tocopherol was 87.0%.

[0038] Verification Example The conditions of Example 1 were used for verification, and the optimal conditions were verified by orthogonal method.

[0039] The effects of using different eluent ratios on step S102 are shown in Tables 1 and 2: Table 1 Table 2

[0040] A comprehensive evaluation was conducted based on Table 2 (CV value, using both yield and purity as indicators), and the results are as follows: Figure 2 As shown. From Figure 2 It can be seen that item 1 scored the highest, followed by item 5.

[0041] As can be seen from Tables 1 and 2, only a specific ratio of cyclohexane-ethanol-n-propanol can separate β-tocopherol (which coexists with α-tocopherol and is very easy to separate subsequently) and γ-tocopherol with high yield and high purity.

[0042] The effects of using different eluent ratios on step S103 are shown in Tables 3 and 4: Table 3

[0043] Table 4

[0044] As can be seen from Tables 3 and 4, specific conditions are required to elute only β-tocopherol with both high yield and purity.

[0045] The effects of eluting components at different time points on step S104 are shown in Table 5: Table 5

[0046] As can be seen from Table 5, collecting FR2-FR7 can achieve a high overall yield and high purity, but the elution fraction in the later stage contains more impurities.

[0047] Under the conditions of Example 1, the second silica gel column was regenerated, and the results are shown in Table 6: Table 6

[0048] As can be seen from Table 6, the second silicone column can be regenerated at least 15 times using the method of this patent.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for the isolation and purification of tocopherol monomers, characterized in that, The method includes the following steps: S101: Concentrate natural vitamin E to increase the mass concentration of tocopherol to 40-65%; S102: The concentrate obtained in step S101 is diluted with cyclohexane and subjected to chromatography using a first silica gel column. The eluent is cyclohexane-ethanol-n-propanol = 100:0.1-0.3:1-2. Fourteen equal elution fractions are obtained and named Fr1-Fr14. The elution fractions Fr3-Fr5, Fr8-Fr9, and Fr11-Fr12 are collected. The elution fractions Fr8-Fr9 and Fr11-Fr12 are concentrated under reduced pressure to obtain γ-tocopherol and δ-tocopherol, respectively. The elution fraction Fr3-Fr5 is concentrated under reduced pressure until the tocopherol concentration reaches above 0.5 g / mL to obtain a first chromatographic concentrate. S103: Chromatography was performed using a second silica gel column. The concentrated solution from the first chromatography was directly loaded onto the column. The eluent was cyclohexane-ethyl acetate = 100:70-85. The eluent was collected and concentrated under reduced pressure to obtain β-tocopherol. S104: The second silica gel column was regenerated with cyclohexane, the eluent was collected, and the α-tocopherol was obtained by vacuum concentration. Both the first silica gel column and the second silica gel column are low-pressure chromatography columns.

2. The method for separating and purifying tocopherol monomers according to claim 1, characterized in that, In step S102, the height-to-diameter ratio of the first silica gel column is 15-25:1, and the particle size of the silica gel used is 100-200 mesh.

3. The method for separating and purifying tocopherol monomers according to claim 1, characterized in that, In step S102, the concentrate is diluted with cyclohexane to a tocopherol concentration of 0.5-2.0 g / mL.

4. The method for separating and purifying tocopherol monomers according to claim 1, characterized in that, In step S102, the flow rate of the eluent is 3-8 column volumes / h, the sample loading volume is 80-150 mg / mL column volume, and one eluent fraction is collected every five minutes.

5. The method for separating and purifying tocopherol monomers according to claim 1, characterized in that, The first silica gel column was regenerated by eluting with 1-3 column volumes of methanol followed by 1-3 column volumes of cyclohexane.

6. The method for separating and purifying tocopherol monomers according to claim 1, characterized in that, The height-to-diameter ratio of the second silica column is 5-10:1, and the particle size of the silica used is 100-200 mesh.

7. The method for separating and purifying tocopherol monomers according to claim 1, characterized in that, In step S103, the flow rate of the eluent is 1-4 column volumes / h, and the sample loading volume is 70-120 mg / mL column volume.

8. The method for separating and purifying tocopherol monomers according to claim 1, characterized in that, In step S104, the second silica gel column is regenerated with 3-5 column volumes of cyclohexane to obtain 10 equal eluent fractions, named FR1-FR10 respectively; the eluent fractions FR2-FR7 are collected and concentrated under reduced pressure to obtain α-tocopherol.

9. The method for separating and purifying tocopherol monomers according to claim 1, characterized in that, In step S102, the eluent is cyclohexane-ethanol-n-propanol = 100:0.2:1; in step S103, the eluent is cyclohexane-ethyl acetate = 100:80.

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