CN110229089B

CN110229089B - Method for separating and purifying high-purity canthaxanthin by using double solvents in combination with medium-pressure liquid chromatography

Info

Publication number: CN110229089B
Application number: CN201910481317.4A
Authority: CN
Inventors: 李笃信; 兰韬; 高叶; 刘嘉雯; 邱飘飘
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2019-06-04
Filing date: 2019-06-04
Publication date: 2021-05-14
Anticipated expiration: 2039-06-04
Also published as: CN110229089A

Abstract

The invention discloses a method for separating and purifying high-purity canthaxanthin by combining a double solvent with a medium-pressure liquid chromatography, which can extract canthaxanthin from a canthaxanthin feed additive by using a single solvent, load the sample to a medium-pressure glass column filled with a silica gel filler, elute the sample isocratically by using a binary mixed solvent, collect high-purity fractions, increase the concentration of a crude sample from 10 percent to more than 99 percent by one step, and stabilize the yield of the purified sample to more than 90 percent. The method has the advantages that the product purity is high, the sample loading amount of the crude sample is large, and the purification cost is greatly saved; the linear flow rate in the chromatographic purification is high, the canthaxanthin can be quickly and efficiently purified, the yield is high, and the time is saved. In addition, the method is simple to operate, stable and reliable, has strong scalability, and is suitable for industrially preparing the double-solvent combined medium-pressure liquid chromatography for separating and purifying the high-purity canthaxanthin.

Description

Method for separating and purifying high-purity canthaxanthin by using double solvents in combination with medium-pressure liquid chromatography

Technical Field

The invention belongs to the field of food safety, and particularly relates to a purification method of a high-purity cantharis yellow reference substance.

Background

The canthaxanthin (or canthaxanthin, CAS number: 514-78-3) is a carotenoid of vitamin A source, and the chemical name is beta-carotene-4, 4' -diketone. As shown in figure 1, cantharis yellow crystal mainly has all-trans structure, and cantharis yellow is dark purple crystal or crystalline powder, has melting point of about 210 deg.C, is unstable to oxygen and light, and needs to be stored in inert gas-shielding container. Canthaxanthin can be dissolved in chloroform (10%), slightly soluble in vegetable oil (0.005%), acetone (0.03%), and insoluble in water, ethanol, and propylene glycol.

Canthaxanthin is commonly used in the food and feed industry as a pigment, and its product has been used in the field of animal and food coloring for more than 40 years to improve poultry skin color and yolk color to increase commercial value of poultry and eggs. Cantharides yellow is also a raw material of vitamin A, and can supplement vitamin A. The natural canthaxanthin is widely existed in crustacean, fowl and algae, has the functions of resisting oxidation and eliminating free radicals, but has little content in organism. With the industrialization of artificially synthesized cantharis yellow, the cantharis yellow is widely applied to industries such as feed, food, chemical industry, medicine and the like. In 1984, the FDA/WHO approved canthaxanthin to be included as a food additive and established quality standards.

At present, canthaxanthin is mainly obtained by four ways, namely natural animal and plant extraction, chemical synthesis, microbial culture and fermentation and beta-carotene oxidation. The natural animal and plant cantharis yellow has limited source and lower content, and the problems of difficult strain screening, complex process, lower extraction efficiency and the like of a biological fermentation method limit the industrial scale production. The chemical synthesis method for preparing canthaxanthin is feasible theoretically, but the canthaxanthin has more isomers, and the canthaxanthin is easy to deteriorate under the photo-thermal oxygen-containing condition, so that the canthaxanthin is not commonly applied to industrial preparation. The microbial culture and fermentation are simpler in synthesis in a laboratory, but the application to industrial production has some technical problems which are difficult to solve.

The current patents for the industrial preparation of canthaxanthin are mainly through the oxidation of beta-carotene. For example, chinese patent publication No. CN 108101817a, chinese patent publication No. CN 1793098A, and chinese patent publication No. CN 105777599a all disclose methods for preparing canthaxanthin by oxidation of β -carotene. However, the above method generally uses a large amount of organic reagents, oxidizing agents, and the like, which are not recyclable, resulting in waste of reagents and low yield. Chinese patent publication No. CN 105777599a discloses the use of halogenated hydrocarbon for extracting canthaxanthin, and because of the toxicity of halogenated hydrocarbon reagents, the content of halogenated hydrocarbon must be strictly ensured not to exceed the maximum range for application in food. The halogenated hydrocarbon reagent has high volatility, but is difficult to remove the halogenated hydrocarbon pollutant in canthaxanthin. Chinese patent publication No. CN 1793098A reports a method for oxidizing beta-carotene by using halate as an oxidizing agent, but the waste of the halate after use is not easy to be treated, and causes a certain pollution to the environment. More importantly, the purity of the canthaxanthin prepared by the beta-carotene oxidation method can not meet the requirements of national standard substances, and the purity of the canthaxanthin samples obtained by the method is less than 90 percent, so the canthaxanthin can not be used as a reference substance for measuring the content in food and feed. Therefore, it is very important to establish a purification method which is feasible theoretically and is suitable for industrial preparation of high-purity canthaxanthin.

Disclosure of Invention

The invention aims to provide a method for separating and purifying high-purity cantharis yellow by combining a double solvent with a medium-pressure liquid chromatography, and solves the problems.

The technical scheme of the invention is as follows:

a method for separating and purifying high-purity canthaxanthin by using a double-solvent combined medium-pressure liquid chromatography is characterized by comprising the following steps of:

(1) extracting cantharis yellow from the raw materials by using a single solvent;

(2) loading the sample to a medium-pressure glass column for chromatography;

(3) isocratic elution with binary solvent;

(4) collecting the target components to obtain the pure product.

Further, the extractant in the step (1) is any one or more of acetonitrile, chloroform, ethyl acetate and acetone.

Further, in the step (2), the ligand of the chromatographic packing is any one or two of phenyl, pentafluorophenyl, pentabromophenyl or C18.

Further, the ligand of the chromatographic filler in the step (2) is a combination of C18 and pentafluorophenyl, and the ratio of C18 to pentafluorophenyl is 2: 8.

further, in the step (2), the average particle size of the chromatographic packing is 15-35 μm, and the pore diameter of the chromatographic packing is

Or

Further, in the step (2), the average particle size of the chromatographic packing is 15 μm, and the pore diameter of the chromatographic packing is

Silica gel matrix filler of (1).

Further, the phase A and the phase B of the binary solvent in the step (3) are any two of methanol, acetonitrile, tetrahydrofuran and ethyl acetate.

Further, in the step (3), the phase A and the phase B of the binary solvent are acetonitrile and tetrahydrofuran, and the mixing ratio is 35: 65.

further, the fraction collecting time period in the step (4) is 1-5 times of column volume of elution.

Further, the fraction collecting time period in the step (4) is 1.5 times to 2.0 times of column volume of elution.

The invention provides a method for separating and purifying high-purity cantharis yellow by combining a double solvent with a medium-pressure liquid chromatography, which has the advantages that:

(1) the invention adopts the mixed filler as the chromatographic stationary phase, more than 99 percent of canthaxanthin can be prepared and purified from 10 percent of crude canthaxanthin, the sample yield after purification is stable at more than 85 percent, and the purity and the yield of the purified sample are far higher than the achievements obtained by the prior art, which is very important for the preparation of reference substances and standard substances;

(2) the invention does not need to be heated, and the cantharis yellow which is not easy to deteriorate can be obtained by vacuum concentration under reduced pressure during concentration and is protected from light during drying, while the cantharis yellow prepared by the existing oxidation method can be prepared only by heating or illumination, thereby greatly increasing the probability of the cantharis yellow deteriorating or obtaining other isomers;

(3) the crude sample to be purified in the invention has large sample loading amount, thus greatly saving the cost, and the existing oxidation methods for preparing canthaxanthin have large amount of oxidant and organic reagent dosage, high cost and serious environmental pollution. Under the condition of increasing concern on environmental protection requirements and body health of production personnel, the reduction of the consumption of the solvent is particularly important;

(4) the chromatographic purification process is rapid, cantharis yellow eluent can be collected in about ten minutes, and adverse effects of target products in the purification process, such as isomer and the like, are better avoided, so that the purity and the yield of a sample are influenced.

Drawings

FIG. 1 shows the molecular structure of canthaxanthin;

FIG. 2 shows a schematic representation of a structure using C18-5 μm-

(4.6 mm. times.250 mm) analytical chromatographic purification pattern of analytical chromatographic column；

FIG. 3 shows the results obtained by using PFP-C18-15 μm-

(15 mm. times.460 mm) medium pressure preparative chromatography purification profile of preparative chromatography column;

FIG. 4 shows the results obtained by using PFP-C18-15 μm-

Chromatogram of continuous preparation of cantharis yellow of (15mm × 460mm) preparative chromatographic column;

FIG. 5 is an analytical chromatogram of a purified canthaxanthin product after preparation and medium-pressure chromatography;

FIG. 6 is a mass spectrum of purified cantharis yellow product after medium pressure chromatography;

FIG. 7 is the hydrogen spectrum of purified cantharis yellow product after medium pressure chromatography.

Detailed Description

The technical scheme of the invention is as follows:

(2) loading the sample to a medium-pressure glass column for chromatography;

(3) isocratic elution with binary solvent;

(4) collecting the target components to obtain the pure product.

According to the chromatographic purification method, the extractant in the step (1) is one or more of acetonitrile, chloroform, ethyl acetate and acetone, and is preferably acetonitrile. The invention achieves the purpose of extracting crude cantharis yellow product from feed additive as much as possible by comparing the solubility of feed-grade cantharis yellow (the purity is 10%) in different organic reagents. The analytical chromatogram of the extracted sample is shown in FIG. 2. The extraction efficiency of each extraction solvent is shown in table 1.

The invention discusses the influence of different ligands, granularity and aperture of the cantharis yellow extracting agent and the chromatographic packing and the selection of the mobile phase on the separation and purification effect of the cantharis yellow through a controlled variable method. The results show that the extraction efficiency of different extractants for cantharis yellow is different and has larger difference. Wherein the efficiency of extracting the acetonitrile in the feed additive is relatively highest.

The different ligands of the chromatographic packing have different influences on the separation effect of canthaxanthin: the four ligands of phenyl, pentafluorophenyl, pentabromophenyl and C18 can achieve better purification effect, the purity of the purified sample reaches 96.83% -99.50%, and the purification yield is 80% -90%: wherein the purification effect of the C18 ligand on canthaxanthin is better than that of other two filler ligands, and the two filler ligands have statistically significant difference. The particle size and pore size of the chromatography packing also have an effect on the separation. It was found that the smaller the particle size of the filler, the higher the degree of separation and the higher the yield. For this reason, in medium pressure preparative chromatography, we selected a filler with a particle size of 15 μm. The aperture of the filler has no great influence on the purity and the yield of the canthaxanthin, but has obvious influence on the single sampling amount. Wherein the pore diameter is

The highest loading of filler (2).

The choice of mobile phase has a major impact on the present invention. In the pairwise matching of organic reagents such as methanol, acetonitrile, tetrahydrofuran, ethyl acetate and the like or in the pairwise matching of methanol, water and acetonitrile, the separation effects of different reagents have obvious differences. The research finds that the mobile phase is methanol-acetonitrile, methanol-tetrahydrofuran or acetonitrile-tetrahydrofuran, the separation effect is better, acetonitrile-tetrahydrofuran (35-65) is preferred, and the purification yield is highest.

The fraction collection time of the invention has higher influence on the purity and the yield, maintains extremely high purity, and greatly reduces the recovery rate of the sample. Therefore, according to the prior experience, the purity of the sample is improved as much as possible while maintaining the recovery rate of 90%, and therefore, the optimum condition is selected to be 1.5 to 2 times the elution volume.

The chromatographic purification method of high-purity canthaxanthin utilizes the relative hydrophobicity and selectivity difference of target products and other impurities in a sample extracted from a chicken feed additive in a specific stationary phase and a specific mobile phase to purify the canthaxanthin. The method is simple and feasible, the sample loading amount of the crude sample is large, the purified sample has high purity and high yield, the purification cost is low, the process is stable and reliable, and the method is suitable for industrially preparing the high-purity cantharis yellow.

The method is suitable for medium-pressure preparative chromatography.

The experimental results of the above method can be seen in fig. 2-7, as shown in fig. 2, peak1 in the figure is the pure canthaxanthin that we want to separate and purify from the preparative chromatography; as shown in fig. 3, peak1 is the collected eluate containing canthaxanthin; as shown in fig. 4, three samples were taken during the preparation period, and the baseline was not flat due to multiple loading, but did not affect the collection of canthaxanthin; as shown in fig. 5, the peak is the pure cantharis yellow product with the purity as high as 99.5%; as shown in FIG. 6, the substance with the nuclear-to-cytoplasmic ratio of 565.4002 is Mylabris to be detected, and other debris peaks are not referred to; as shown in fig. 7, it can be seen from the integration of hydrogen in the figure and comparison with the hydrogen spectrum of canthaxanthin in the literature: the substance is the pure cantharis yellow prepared by the invention.

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. The invention is not limited to the embodiments listed but also comprises any other known variations within the scope of the invention as claimed.

Reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic may be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

The term "reverse phase silica gel" is a silicon oxide material coated on its surface with a hydrophobic matrix, wherein the hydrophobic matrix may be an alkane; the coating is that Si-OH groups on the surface of the silica gel are bonded with different functional groups to form chromatographic packing suitable for different separation modes; silica gels can generally be chemically modified to prepare silica gel-bonded phases by three routes: coating method, integral modification method and chemical modification method of surface Si-OH. In the present invention, the term "reverse phase silica gel" has the same meaning as the term "advanced silica gel support", and the two may be interchanged; the term "preparative chromatography" is to be understood as the preparation of a pure product on an industrial scale.

1. Test materials and instruments

Acetonitrile (CH)₃CN, preparative chromatography grade) from high purity reagents ltd, preparative chromatography columns (15mm × 460mm), packing and preparative hplc were from shivikon mass chromatography separation and purification ltd.

2. The sample purity detection method comprises the following steps:

the crude sample and the purified collected sample were subjected to detection by reverse phase chromatography using ODS-C18-5 μm column (4.6 mm. times.250 mm). The flow rate was 1mL/min, the column temperature was 25 ℃ and the detection wavelength was 470 nm. Isocratic elution for 20 min. The main peak with the retention time of 12-13min is the target product. The purity was calculated using area normalization.

The extraction method of cantharis yellow in the feed additive is prepared and implemented.

Weighing an appropriate amount of 100g of the chicken feed additive, dividing into four equal parts, respectively adding 200mL of acetonitrile, acetone, ethyl acetate and chloroform, stirring for 60min under a magnetic stirrer, filtering with a 0.45 μm filter membrane, and collecting the filtrate to obtain cantharis yellow extract.

Example 1

Research on influence of different extractants on extraction efficiency of canthaxanthin in feed additive

The experimental method comprises the following steps: the crude sample is as above, 10% feed grade cantharis yellow. The chromatographic purification method adopts (C18-15 mu m-

) On a medium pressure preparative chromatography column (15 mm. times.460 mm) at a packing pressure of 3 MPa; the mobile phase for elution is methanol; the elution flow rate is 18 mL/min; the detection wavelength was 470 nm.

The implementation steps are as follows:

(1) extracting the feed additive by using the extracting solution;

(2) the crude cantharis yellow sample 10mL extracted was loaded on the column and then the column was equilibrated with equilibration solution until baseline was stable.

(3) The mobile phase was methanol and elution was carried out with a monitoring wavelength of 470 nm.

(4) Collecting for 12-13 min;

TABLE 1 Effect of different extraction solvents on purity and yield

As can be seen from table 1, there is not much difference in purity of canthaxanthin extracted by four solvents, but the difference in yield is great. Acetonitrile is used as an extraction solvent, and the yield is highest. Therefore, acetonitrile is selected as an extraction solvent to extract canthaxanthin in the feed additive.

Example 2

Research on influence of different granularity of chromatography filler on separation effect of canthaxanthin in feed additive

The experimental method comprises the following steps: the crude sample is as above, 10% feed grade cantharis yellow. The chromatographic purification method is carried out on a medium-pressure preparative chromatographic column (15mm multiplied by 460mm), and the filling pressure is 3 MPa; the mobile phase for elution is methanol; the elution flow rate is 18 mL/min; the detection wavelength was 470 nm.

The implementation steps are as follows:

(1) extracting the feed additive by adopting acetonitrile;

(2) the 10mL extracted crude cantharis yellow sample is loaded to a medium pressure glass column, and then the column is balanced by a balance liquid until the baseline is stable. Filling the medium-pressure glass column respectively:

C18-15μm-

C18-20μm-

C18-20μm-

(4) Collecting for 12-13 min;

the test results are shown in Table 2

TABLE 2 Effect of different filler particle sizes on purity and yield

The experimental result shows that the smaller the filler granularity is, the higher the purity and yield of the purified sample are. As can be seen from the table, the filler with a particle size of 15 μm has the best purification effect on canthaxanthin, which is shown in that the purity of the sample after purification is high, 99.31%, and the purification yield is relatively highest. Therefore, the filler with the particle size of 15 μm is preferably used as the filler for purifying canthaxanthin.

Example 3

Research on influence of different pore diameters of chromatographic filler on separation and purification of canthaxanthin in feed additive

The experimental method comprises the following steps: the crude sample is canthaxanthin crude sample solution extracted from acetonitrile: the chromatographic purification method adopts the same silica gel filler and the same particle size (C18-15 μm) but different pore diameters

The packing of (2) was carried out on a preparative column (15 mm. times.460 mm) at a packing pressure of 3 MPa; eluting with mobile phase methanol at flow rate of 18 mL/min; the detection wavelength is 470nm

The implementation steps are as follows:

(1) extracting the feed additive by adopting acetonitrile;

C18-15μm-

C18-15μm-

C18-15μm-

C18-15μm-

(4) Collecting for 12-13 min;

the test results are shown in Table 3

TABLE 3 Effect of different filler pore sizes on purity and yield

The experimental result shows that the smaller the pore size of the filler is, the larger the single sample loading amount of the filler is. As can be seen from the table, the purification effect of the filler with four pore diameters on canthaxanthin is similar, but the pore diameter is

The single-time sample loading amount of the filling is higher, which results in that the pure cantharides yellow product obtained at a time is also higher. Under the condition of preparing the same amount of canthaxanthin pure product, the cost of the consumed reagent is the lowest. Therefore, the preferred pore diameter is

The filler is used as the filler for purifying cantharis yellow.

Example 4

Investigation of the Effect of ligand Filler on the separation Effect

The implementation steps are as follows:

(1) extracting the feed additive by adopting acetonitrile;

C4-15μm-

C8-15μm-

C18-15μm-

phenyl-15 μm-

Pentafluorophenyl 15 μm-

Pentabromophenyl 15 μm-

pentafluorophenyl-C18-15 μm-

pentabromophenyl-C18-15 μm-

pentabromophenyl-C8-15 μm-

(4) Collecting for 12-13 min;

the test results are shown in Table 4

TABLE 4 Effect of different fillers on purity and yield

The experimental results are as follows: as can be seen from the table, pentafluorophenyl-C18-5 μm-

The mixed filler has the best purification effect on canthaxanthin, which is shown in that the purity of a purified sample is higher and reaches 99.16%, and the purification yield is relatively highest. Thus, pentafluorophenyl-C18-5 μm-

The filler is used as the filler for purifying cantharis yellow.

Example 5

Research on the influence of different eluents on the separation and purification of cantharidin in feed additive

The experimental method comprises the following steps: the crude sample is as above, 10% feed grade cantharis yellow. The chromatographic purification method is carried out on a medium-pressure preparative chromatographic column (15mm multiplied by 460mm), and the filling pressure is 3 MPa; the elution flow rate is 18 mL/min; the detection wavelength was 470 nm.

The implementation steps are as follows:

(1) extracting the feed additive by adopting acetonitrile;

(2) the 10mL extracted crude cantharis yellow sample is loaded to a medium pressure glass column, and then the column is balanced by a balance liquid until the baseline is stable. Filling the medium-pressure glass column: c18-15 μm-

(3) Elution was carried out using the mobile phase, monitoring the wavelength 470 nm.

The mobile phases are respectively: methanol: acetonitrile, methanol: tetrahydrofuran, tetrahydrofuran: acetonitrile, acetonitrile: water, ethyl acetate: acetonitrile

(4) Collecting for 12-13 min;

the test results are shown in Table 5

TABLE 5 influence of the flow relative purity and yield for different elutions

The experimental results are as follows: as can be seen from the table, the selection of the mobile phase has a great influence on the purification and yield of canthaxanthin. The separation effect of different reagents is found to have obvious difference in the two-in-one ratio of organic reagents such as methanol, acetonitrile, tetrahydrofuran, ethyl acetate and the like or the two-in-one ratio of methanol, water and acetonitrile. The research finds that the mobile phase is methanol-acetonitrile, methanol-tetrahydrofuran or acetonitrile-tetrahydrofuran, the separation effect is better, and the mobile phase is preferably methanol-acetonitrile (50: 50), and the purification yield is highest.

Example 6

The comparison of the main process parameters and the purification effect between the present invention and the related Chinese patents is studied and shown in Table 6

In conclusion, the method for separating and purifying the high-purity cantharis yellow by combining the double solvents with the medium-pressure liquid chromatography provided by the invention utilizes the medium-pressure liquid chromatography to prepare the cantharis yellow pure product with the purity of more than 99%. The organic reagent used in the invention, such as acetonitrile and the like, can be recovered by adopting a technical means, and cannot cause environmental pollution and reagent waste. Secondly, the chromatographic purification method does not need the conditions of photo-thermal and oxygen-containing which are easy to cause the cantharis to be yellow-modified, and has mild environment and simple and convenient operation. The purity of canthaxanthin separated by the chromatography is greatly increased, the content of canthaxanthin is improved to more than 99 percent from the content of 2.5 percent, 10 percent and the like which are common in the market, and the canthaxanthin is more suitable to be used as a standard substance and a reference substance of canthaxanthin analytes in food and medicine industries. The method can rapidly increase the concentration of the crude sample from 10% to more than 99%, and the sample yield after purification is more than 95%, has strong scalability, and is suitable for being used as a chromatographic purification method for industrialized purification of canthaxanthin.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims

1. A method for separating and purifying canthaxanthin by combining two solvents and medium-pressure liquid chromatography is characterized by comprising the following steps:

(1) extracting cantharis yellow from raw materials by using a single solvent, wherein the single solvent is acetonitrile, and the raw materials are 10% of feed-grade cantharis yellow;

(2) loading the mixture to a medium-pressure glass column for chromatography, wherein ligands of chromatography packing are a combination of C18 and pentafluorophenyl, and the ratio of C18 to pentafluorophenyl is 2: 8, the chromatographic filler has an average particle size of 15 μm, the chromatographic filler has a pore diameter of 60 a, and the chromatographic filler is a silica gel matrix filler;

(3) isocratic elution is carried out by using a binary solvent, wherein the phase A of the binary solvent is acetonitrile, the phase B of the binary solvent is tetrahydrofuran, and the mixing ratio is 35: 65;

(4) and collecting the target component to obtain a pure product, wherein the fraction collecting time period is 1-5 times of the column volume of the elution.

2. The method for separating and purifying canthaxanthin by using two-solvent combined medium-pressure liquid chromatography as claimed in claim 1, wherein the method comprises the following steps: and (4) collecting fractions in the step (4) for a time period of eluting 1.5-2.0 times of column volume.