CN114377432A - Method for separating haematococcus pluvialis extract by supercritical fluid chromatography - Google Patents

Method for separating haematococcus pluvialis extract by supercritical fluid chromatography Download PDF

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CN114377432A
CN114377432A CN202111570709.1A CN202111570709A CN114377432A CN 114377432 A CN114377432 A CN 114377432A CN 202111570709 A CN202111570709 A CN 202111570709A CN 114377432 A CN114377432 A CN 114377432A
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carbon dioxide
volume fraction
silica gel
haematococcus pluvialis
supercritical carbon
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CN114377432B (en
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赵建强
祁威
李枝芳
金海蛟
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Jiangsu Hanbon Science and Technology Co Ltd
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/40—Selective adsorption, e.g. chromatography characterised by the separation mechanism using supercritical fluid as mobile phase or eluent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C403/00—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone
    • C07C403/24—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone having side-chains substituted by six-membered non-aromatic rings, e.g. beta-carotene
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
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Abstract

The invention belongs to the technical field of substance purification, and provides a method for separating haematococcus pluvialis extract by supercritical fluid chromatography. The method of the invention dissolves the haematococcus pluvialis extract in a low-polarity solvent, and adopts a supercritical fluid chromatography method to carry out separation on a stationary phase; the mobile phase system is a mixture of supercritical carbon dioxide and lower alcohol, and the volume ratio of the mixture is 99-50: 1-50; the modified silica gel chromatographic column is filled with silica gel with pyridine, amino, phenyl, cyano, diol group, C18C, C18P or C18H bonded on the surface, and can well realize the separation of astaxanthin with different configurations in haematococcus pluvialis extract.

Description

Method for separating haematococcus pluvialis extract by supercritical fluid chromatography
Technical Field
The invention relates to the technical field of substance purification, in particular to a method for separating haematococcus pluvialis extract by supercritical fluid chromatography.
Background
Astaxanthin is widely found in the biological world, particularly in feathers of aquatic animals such as shrimps, crabs, fish and birds, and plays a color-developing role. Astaxanthin is a chain-breaking antioxidant, has strong oxidation resistance, can remove nitrogen dioxide, sulfide, disulfide, etc., can reduce lipid peroxidation, and effectively inhibit lipid peroxidation caused by free radicals. Meanwhile, the traditional Chinese medicine composition has physiological effects in various aspects such as inhibiting tumorigenesis, enhancing immunity, eliminating free radicals in vivo and the like, has a good treatment effect on skin cancer caused by ultraviolet rays, has a prevention and treatment effect on eye diseases caused by diabetes, and has a wide application prospect in the aspects of health care products, medicines, cosmetics, food additives, aquaculture and the like.
The natural astaxanthin content of the haematococcus pluvialis extract is relatively high and reaches 10-30%. But astaxanthin has a plurality of isomers with different configurations, and the astaxanthin with different configurations has different oxidation resistance. Meanwhile, due to the similar structures of the astaxanthin with different configurations, the common high performance liquid chromatography method cannot well separate the astaxanthin with different configurations.
Disclosure of Invention
In view of the above, the present invention provides a method for separating haematococcus pluvialis extract by supercritical fluid chromatography. The method provided by the invention can separate astaxanthin with different configurations.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for separating haematococcus pluvialis extract by supercritical fluid chromatography, which comprises the following steps:
dissolving Haematococcus pluvialis extract in a low-polarity solvent to obtain a Haematococcus pluvialis extract solution;
performing supercritical fluid chromatographic separation on the haematococcus pluvialis extract solution;
the parameters of the supercritical fluid chromatographic separation include:
the chromatographic column comprises a modified silica gel chromatographic column;
the filler of the modified silica gel chromatographic column is silica gel with the surface bonded with pyridine, amino, phenyl, cyano, diol group, C18C, C18P or C18H;
the mobile phase system comprises a mobile phase A and a mobile phase B; the mobile phase A is supercritical carbon dioxide, and the mobile phase B is lower alcohol;
in the process of gradient elution, the volume ratio of the mobile phase A to the mobile phase B is 99-50: 1 to 50. Preferably, the low polarity solvent comprises ethyl acetate or dichloromethane.
Preferably, the concentration of the haematococcus pluvialis extract solution is 30-200 mg/mL.
Preferably, the lower alcohol comprises methanol or ethanol.
Preferably, the parameters of the supercritical fluid chromatographic separation further include: the detector is a PAD detector; the detection wavelength of the PAD detector is 472 nm.
Preferably, when the packing material of the modified silica gel chromatographic column is silica gel with C18C bonded on the surface, the procedure of gradient elution is as follows:
0 min: the volume fraction of the supercritical carbon dioxide is 90 percent, and the volume fraction of the ethanol is 10 percent;
0-15 min: the volume fraction of the supercritical carbon dioxide is maintained to be 90 percent;
15-30 min; the volume fraction of the supercritical carbon dioxide is changed from 90% to 50% at a constant speed.
Preferably, when the packing material of the modified silica gel chromatographic column is silica gel with C18C bonded on the surface, the procedure of gradient elution is as follows:
0 min: the volume fraction of the supercritical carbon dioxide is 99 percent, and the volume fraction of the ethanol is 1 percent;
0-50 min: the volume fraction of the supercritical carbon dioxide is maintained at 99 percent;
50-51 min: the volume fraction of the supercritical carbon dioxide is changed from 90% to 50% at a constant speed;
51-70 min: the volume fraction of supercritical carbon dioxide was maintained at 50%.
Preferably, when the packing material of the modified silica gel chromatographic column is silica gel with C18P bonded on the surface, the procedure of gradient elution is as follows:
0 min: the volume fraction of the supercritical carbon dioxide is 97 percent, and the volume fraction of the ethanol is 3 percent;
0-30 min: the volume fraction of supercritical carbon dioxide was maintained at 97%.
Preferably, when the filler of the modified silica gel chromatographic column is silica gel with amino groups bonded on the surface, the procedure of gradient elution is as follows:
0 min: the volume fraction of the supercritical carbon dioxide is 92 percent, and the volume fraction of the ethanol is 8 percent;
0-20 min: the volume fraction of the supercritical carbon dioxide is maintained at 92%;
20-25 min; the volume fraction of the supercritical carbon dioxide is changed from 92% to 50% at a constant speed.
Preferably, the dimensions of the chromatography column comprise 4.6 × 250mm, 10 × 250mm, 20 × 250mm or 50 × 250 mm.
The invention provides a method for separating haematococcus pluvialis extract by supercritical fluid chromatography, which comprises the following steps: dissolving Haematococcus pluvialis extract in a low-polarity solvent to obtain a Haematococcus pluvialis extract solution; performing supercritical fluid chromatographic separation on the haematococcus pluvialis extract solution; the parameters of the supercritical fluid chromatographic separation include: the chromatographic column comprises a modified silica gel chromatographic column; the filler of the modified silica gel chromatographic column is silica gel with the surface bonded with pyridine, amino, phenyl, cyano, diol group, C18C, C18P or C18H; the mobile phase system comprises a mobile phase A and a mobile phase B; the mobile phase A is supercritical carbon dioxide, and the mobile phase B is lower alcohol; in the process of gradient elution, the volume ratio of the mobile phase A to the mobile phase B is 99-50: 1 to 50. The method of the invention dissolves the haematococcus pluvialis extract in a low-polarity solvent, and adopts a supercritical fluid chromatography method to carry out separation on a stationary phase; the mobile phase system is a mixture of supercritical carbon dioxide and lower alcohol, and the volume ratio of the mixture is 99-50: 1-50; the modified silica gel chromatographic column is filled with silica gel with pyridine, amino, phenyl, cyano, diol group, C18C, C18P or C18H bonded on the surface, and can well realize the separation of astaxanthin with different configurations in haematococcus pluvialis extract.
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FIG. 1 is a spectrum of Haematococcus pluvialis extract solution of example 1 obtained by supercritical fluid chromatography;
FIG. 2 is a spectrum of Haematococcus pluvialis extract solution of example 2 obtained by supercritical fluid chromatography;
FIG. 3 is a spectrum of Haematococcus pluvialis extract solution of example 3 obtained by supercritical fluid chromatography;
FIG. 4 is a spectrum of Haematococcus pluvialis extract solution of example 4 obtained by supercritical fluid chromatography;
FIG. 5 is a chromatogram of Haematococcus pluvialis extract solution of example 5 obtained using supercritical fluid chromatography;
FIG. 6 is a chromatogram of Haematococcus pluvialis extract solution of example 6 obtained using supercritical fluid chromatography;
FIG. 7 is a spectrum obtained by separating Haematococcus pluvialis extract solution of comparative example 1 by high performance liquid chromatography;
FIG. 8 is a spectrum of the Haematococcus pluvialis extract solution of comparative example 2 separated by high performance liquid chromatography.
Detailed Description
The invention provides a method for separating haematococcus pluvialis extract by supercritical fluid chromatography, which comprises the following steps:
dissolving Haematococcus pluvialis extract in a low-polarity solvent to obtain a Haematococcus pluvialis extract solution;
performing supercritical fluid chromatographic separation on the haematococcus pluvialis extract solution;
the parameters of the supercritical fluid chromatographic separation include:
the chromatographic column comprises a modified silica gel chromatographic column;
the filler of the modified silica gel chromatographic column is silica gel with the surface bonded with pyridine, amino, phenyl, cyano, diol group, C18C, C18P or C18H;
the mobile phase system comprises a mobile phase A and a mobile phase B; the mobile phase A is supercritical carbon dioxide, and the mobile phase B is lower alcohol;
in the process of gradient elution, the volume ratio of the mobile phase A to the mobile phase B is 99-50: 1 to 50.
In the present invention, the starting materials used in the present invention are preferably commercially available products unless otherwise specified.
The haematococcus pluvialis extract is dissolved in a low-polarity solvent to obtain a haematococcus pluvialis extract solution.
In the invention, the mass content of astaxanthin in the haematococcus pluvialis extract is preferably 10-30%. In the invention, the haematococcus pluvialis extract is preferably a product extracted from haematococcus pluvialis, and the product is subjected to normal phase silica gel column enrichment and partial impurity removal, and then sequentially subjected to the following steps of: 1.5 and the volume ratio is 3: 2, eluting with n-hexane-ethyl acetate, and collecting the eluate at a volume ratio of 3: 2 n-hexane-ethyl acetate elution fraction.
In the present invention, the low-polarity solvent preferably includes ethyl acetate or dichloromethane, and more preferably ethyl acetate. In the invention, the concentration of the haematococcus pluvialis extract solution is preferably 30-200 mg/mL.
After the haematococcus pluvialis extract solution is obtained, the supercritical fluid chromatographic separation is carried out on the haematococcus pluvialis extract solution.
In the present invention, the parameters of the supercritical fluid chromatographic separation include:
the chromatographic column comprises a modified silica gel chromatographic column;
the filler of the modified silica gel chromatographic column is silica gel with the surface bonded with pyridine, amino, phenyl, cyano, diol group, C18C, C18P or C18H; further preferred is a silica gel having a C18C, C18P or amino group bonded to the surface thereof, more preferred is a silica gel having a C18C or amino group bonded to the surface thereof, and most preferred is a silica gel having an amino group bonded to the surface thereof.
The mobile phase system comprises a mobile phase A and a mobile phase B; the mobile phase A is supercritical carbon dioxide; the temperature of the supercritical carbon dioxide when the supercritical carbon dioxide passes through the pump is 5-10 ℃, and the density of the supercritical carbon dioxide is 890-950 kg/m3(ii) a The mobile phase B is lower alcohol; the lower alcohol preferably includes methanol or ethanol, and more preferably ethanol.
In the process of gradient elution, the volume ratio of the mobile phase A to the mobile phase B is 99-50: 1 to 50.
In the present invention, when the packing of the modified silica gel chromatographic column is silica gel with a surface bonded with C18C, the gradient elution procedure is preferably as follows:
0 min: the volume fraction of the supercritical carbon dioxide is 90 percent, and the volume fraction of the ethanol is 10 percent;
0-15 min: the volume fraction of the supercritical carbon dioxide is maintained to be 90 percent;
15-30 min; the volume fraction of the supercritical carbon dioxide is changed from 90% to 50% at a constant speed.
In the present invention, when the packing of the modified silica gel chromatographic column is silica gel with a surface bonded with C18C, the gradient elution procedure is preferably as follows:
0 min: the volume fraction of the supercritical carbon dioxide is 99 percent, and the volume fraction of the ethanol is 1 percent;
0-50 min: the volume fraction of the supercritical carbon dioxide is maintained at 99 percent;
50-51 min: the volume fraction of the supercritical carbon dioxide is changed from 90% to 50% at a constant speed;
51-70 min: the volume fraction of supercritical carbon dioxide was maintained at 50%.
In the present invention, when the packing of the modified silica gel chromatographic column is silica gel with a surface bonded with C18P, the gradient elution procedure is preferably as follows:
0 min: the volume fraction of the supercritical carbon dioxide is 97 percent, and the volume fraction of the ethanol is 3 percent;
0-30 min: the volume fraction of supercritical carbon dioxide was maintained at 97%.
In the present invention, when the filler of the modified silica gel chromatographic column is silica gel with amino groups bonded on the surface, the gradient elution procedure is preferably as follows:
0 min: the volume fraction of the supercritical carbon dioxide is 92 percent, and the volume fraction of the ethanol is 8 percent;
0-20 min: the volume fraction of the supercritical carbon dioxide is maintained at 92%;
20-25 min; the volume fraction of the supercritical carbon dioxide is changed from 92% to 50% at a constant speed.
In the present invention, the size of the chromatography column preferably comprises 4.6 × 250mm, 10 × 250mm, 20 × 250mm or 50 × 250 mm.
In the present invention, when the size of the column is preferably 4.6X 250 mm; the flow rate of the mobile phase system is preferably 2mL/min, and the sample introduction volume is preferably 1-5 muL, and more preferably 2 muL.
In the invention, when the size of the chromatographic column is preferably 10 × 250mm, the flow rate of the mobile phase system is preferably 10mL/min, and the injection volume is preferably 5-20 μ L, and more preferably 5 μ L.
In the present invention, when the size of the column is preferably 20X 250 mm; the flow rate of the mobile phase system is preferably 40mL/min, and the sample introduction volume is preferably 10-30 muL, and more preferably 20 muL.
In the present invention, when the size of the column is preferably 50X 250 mm; the flow rate of the mobile phase system is preferably 250mL/min, and the injection volume is preferably 40-60 muL, and more preferably 50 muL.
In the present invention, the column temperature of the chromatographic column for supercritical fluid chromatography is preferably 30 to 35 ℃, and more preferably 35 ℃.
In the present invention, the back pressure of the supercritical fluid chromatographic separation is preferably 12 MPa.
In the present invention, the parameters of the supercritical fluid chromatographic separation further include: the detector is preferably a PAD detector; the detection wavelength of the PAD detector is preferably 472 nm.
The following examples are provided to illustrate the method of separating Haematococcus pluvialis extract by supercritical fluid chromatography, but they should not be construed as limiting the scope of the present invention.
Example 1
The chromatograph is a supercritical fluid chromatograph of Jiangsu Hanbang science and technology Limited, the specification of a chromatographic column is 4.6 multiplied by 250mm, the flow rate of a mobile phase system is 2mL/min, the back pressure is set to be 12MPa, the concentration of the haematococcus pluvialis extract ethyl acetate solution is 30mg/mL (the content of astaxanthin in the haematococcus pluvialis extract is 30%), the sample injection volume is 2 mu L, and the detection wavelength is 472 nm.
Table 1 shows the conditions for separating astaxanthin from Haematococcus pluvialis extract solution by Supercritical Fluid Chromatography (SFC) in example 1.
TABLE 1 SFC separation conditions in example 1
Figure BDA0003423605510000061
Figure BDA0003423605510000071
The concrete steps corresponding to the embodiment 1 are as follows: at present, haematococcus pluvialis extract with 30% of astaxanthin content is dissolved in ethyl acetate, ultrasonic dissolution is carried out, a solution with the concentration of 30mg/mL is prepared, on a supercritical fluid chromatograph of Jiangsu Hanbang science and technology Limited company, Nucifera C18C 5 mu m 4.6 multiplied by 250mm is selected as a chromatographic column, the volume ratio, the total flow rate, the back pressure and the temperature of the chromatographic column in a flowing phase are controlled according to the table 1, meanwhile, the temperature of carbon dioxide entering a pump is ensured to be 5-10 ℃, the ethyl acetate solution of the haematococcus pluvialis extract is separated, and the separation effect graph is shown in figure 1. As can be seen from fig. 1: the chromatographic column taking Nucifera C18C as a stationary phase can separate six astaxanthin with different configurations in the haematococcus pluvialis extract, and has better separation effect.
Example 2
The chromatograph selected is a supercritical fluid chromatograph of Jiangsu Hanbang science and technology Limited, the specification of a chromatographic column is 10 multiplied by 250mm, the flow rate of a mobile phase system is 10mL/min, the back pressure is set to be 12MPa, the concentration of an ethyl acetate solution of the haematococcus pluvialis extract is 200mg/mL (the mass content of astaxanthin in the haematococcus pluvialis extract is 10%), the sample injection volume is 5 mu L, and the detection wavelength is 472 nm.
Table 2 shows the conditions for separating astaxanthin from Haematococcus pluvialis extract solution by Supercritical Fluid Chromatography (SFC) in example 2.
TABLE 2 SFC separation conditions in example 2
Figure BDA0003423605510000072
Figure BDA0003423605510000081
The concrete steps corresponding to the embodiment 2 are as follows: dissolving haematococcus pluvialis extract with the astaxanthin mass content of 10% in ethyl acetate, performing ultrasonic assisted dissolution to prepare 200mg/mL solution, selecting Nucifera C18C 5 mu m 10 multiplied by 250mm as a chromatographic column on a supercritical fluid chromatograph of Jiangsu Hanbang science and technology Limited, controlling the volume ratio, total flow rate, back pressure and chromatographic column temperature of supercritical carbon dioxide and ethanol in a mobile phase according to table 2, and simultaneously ensuring that the temperature of the carbon dioxide entering a pump is 5-10 ℃. The separation effect of the ethyl acetate solution of haematococcus pluvialis extract is shown in figure 2. As can be seen from fig. 2: the chromatographic column with the stationary phase of Nucifera C18C is most suitable for separating astaxanthin with different configurations, a semi-preparative chromatographic column with the diameter of 10 multiplied by 250mm is used for carrying out preparation experiments, and the preparation effect is better as shown in the figure, and the method can be used as a method for preparing a large amount of astaxanthin.
Example 3
The chromatograph selected is a supercritical fluid chromatograph of Jiangsu Hanbang science and technology Limited, the specification of a chromatographic column is 4.6 multiplied by 250mm, the flow rate of a mobile phase system is 2mL/min, the back pressure is set to be 12MPa, the concentration of an ethyl acetate solution of the haematococcus pluvialis extract is 30mg/mL (the mass content of astaxanthin in the haematococcus pluvialis extract is 30%), the sample injection volume is 2 mu L, and the detection wavelength is 472 nm.
Table 3 shows the conditions for separating astaxanthin from Haematococcus pluvialis extract solution by Supercritical Fluid Chromatography (SFC) in example 3.
TABLE 3 SFC separation conditions in example 3
Figure BDA0003423605510000082
The embodiment 3 comprises the following specific steps: dissolving haematococcus pluvialis extract with the astaxanthin mass content of 30% in ethyl acetate, performing ultrasonic assisted dissolution to prepare a solution with the concentration of 30mg/mL, selecting Nucifera C18P 5 mu m 4.6 multiplied by 250mm as a chromatographic column on a supercritical fluid chromatograph of Jiangsu Hanbang science and technology Limited, and controlling the volume ratio, total flow rate, back pressure and chromatographic column temperature of supercritical carbon dioxide and ethanol in a flowing phase according to a table 3, and simultaneously ensuring that the temperature of the carbon dioxide entering a pump is 5-10 ℃; the separation effect of the ethyl acetate solution of haematococcus pluvialis extract is shown in figure 3. As can be seen from fig. 3: the chromatographic column with the stationary phase of Nucifera C18P has good separation effect on astaxanthin with the configuration of No. 3 and No. 4.
Example 4
The chromatograph selected is a supercritical fluid chromatograph of Jiangsu Hanbang science and technology Limited, the specification of the chromatographic column is 10 multiplied by 250mm, the flow rate of a mobile phase system is 10mL/min, the back pressure is set to be 12MPa, the concentration of the haematococcus pluvialis extract ethyl acetate solution is 200mg/mL (purity is 10%), the sample injection volume is 5 mu L, and the detection wavelength is 472 nm.
Table 4 shows the conditions for separating astaxanthin from Haematococcus pluvialis extract solution by Supercritical Fluid Chromatography (SFC) in example 4.
Table 4 SFC separation conditions in example 4
Figure BDA0003423605510000091
The embodiment 4 comprises the following specific steps: dissolving Haematococcus pluvialis extract containing 10% astaxanthin by weight in ethyl acetate, performing ultrasonic assisted dissolution to obtain 200mg/mL solution, and selecting Nucifera NH on supercritical fluid chromatograph of Jiangsu Hanbang science and technology Limited2Taking the carbon dioxide with the diameter of 5 mu m and the diameter of 10 multiplied by 250mm as a chromatographic column, controlling the volume ratio, total flow rate, back pressure and chromatographic column temperature of the supercritical carbon dioxide and ethanol in the mobile phase according to the table 4, and simultaneously ensuring that the temperature of the carbon dioxide entering a pump is 5-10 ℃; the separation effect of the ethyl acetate solution of haematococcus pluvialis extract is shown in figure 4. As can be seen from fig. 4: the stationary phase is NuciferaNH2The column of (a) can separate the most material.
Example 5
The chromatograph selected is a supercritical fluid chromatograph of Jiangsu Hanbang science and technology Limited, the specification of the chromatographic column is 20 multiplied by 250mm, the flow rate of a mobile phase system is 40mL/min, the back pressure is set to be 12MPa, the concentration of the haematococcus pluvialis extract ethyl acetate solution is 200mg/mL (purity is 10%), the sample injection volume is 20 mu L, and the detection wavelength is 472 nm.
Table 5 shows the conditions for separating astaxanthin from Haematococcus pluvialis extract solution by Supercritical Fluid Chromatography (SFC) in example 5.
TABLE 5 SFC separation conditions in example 5
Figure BDA0003423605510000101
The concrete steps corresponding to the embodiment 5 are as follows: dissolving Haematococcus pluvialis extract containing 10% astaxanthin by weight in ethyl acetate, performing ultrasonic assisted dissolution to obtain 200mg/mL solution, and selecting Nucifera NH on supercritical fluid chromatograph of Jiangsu Hanbang science and technology Limited2Taking 10-micron 20 multiplied by 250mm as a chromatographic column, controlling the volume ratio, total flow rate, back pressure and chromatographic column temperature of supercritical carbon dioxide and ethanol in a mobile phase according to table 5, and simultaneously ensuring that the temperature of carbon dioxide entering a pump is 5-10 ℃; the separation effect of the ethyl acetate solution of haematococcus pluvialis extract is shown in figure 5. As can be seen from fig. 5: the stationary phase is NuciferaNH2The column of (a) can separate the most material.
Example 6
The chromatograph selected is a supercritical fluid chromatograph of Jiangsu Hanbang science and technology Limited, the specification of the chromatographic column is 50 multiplied by 250mm, the flow rate of a mobile phase system is 250mL/min, the back pressure is set to be 12MPa, the concentration of the haematococcus pluvialis extract ethyl acetate solution is 200mg/mL (purity is 10%), the sample injection volume is 50 mu L, and the detection wavelength is 472 nm.
Table 6 shows the conditions for separating astaxanthin from Haematococcus pluvialis extract solution by Supercritical Fluid Chromatography (SFC) in example 6.
TABLE 6 SFC separation conditions in example 6
Figure BDA0003423605510000111
The concrete steps corresponding to the embodiment 6 are as follows: dissolving Haematococcus pluvialis extract containing 10% astaxanthin by weight in ethyl acetate, performing ultrasonic assisted dissolution to obtain 200mg/mL solution, and selecting Nucifera NH on supercritical fluid chromatograph of Jiangsu Hanbang science and technology Limited2Taking 10-micron 50 multiplied by 250mm as a chromatographic column, controlling the volume ratio, total flow rate, back pressure and chromatographic column temperature of supercritical carbon dioxide and ethanol in a mobile phase according to table 6, and simultaneously ensuring that the temperature of carbon dioxide entering a pump is 5-10 ℃; the separation effect of the ethyl acetate solution of haematococcus pluvialis extract is shown in figure 6. As can be seen from fig. 6: the stationary phase is NuciferaNH2The column of (a) can separate the most material.
Comparative example 1
The chromatograph selected is a high performance liquid chromatograph of Jiangsu Hanbang science and technology Limited, the specification of the chromatographic column is 10 multiplied by 250mm, the flow rate of a mobile phase system is 2mL/min, the concentration of the haematococcus pluvialis extract is 200mg/mL (the mass content of astaxanthin in the haematococcus pluvialis extract is 10%), the sample injection volume is 1 mu L, and the detection wavelength is 472 nm.
Table 7 shows the conditions for separating astaxanthin from the Haematococcus pluvialis extract solution in comparative example 1 by high performance liquid chromatography.
TABLE 7 HPLC SEPARATION CONDITIONS OF COMPARATIVE EXAMPLE 1
Figure BDA0003423605510000112
Figure BDA0003423605510000121
The specific steps corresponding to comparative example 1 are: a haematococcus pluvialis extract containing 10% by mass of astaxanthin is dissolved in ethyl acetate, ultrasonic assisted dissolution is carried out, a solution with the concentration of 200mg/mL is prepared, Nucifera C18M 5 with the particle size of 4.6 multiplied by 250mm is selected as a chromatographic column on a high performance liquid chromatograph of Hanbang science and technology Limited, Jiangsu, and the volume ratio, the total flow rate and the column temperature of the chromatographic column in a mobile phase A and B are controlled according to the table 7. The separation effect of the ethyl acetate solution of haematococcus pluvialis extract is shown in figure 7. As can be seen from fig. 7: when the separation is carried out in high performance liquid chromatography by using a Nucifera C18M chromatographic column, the separation degree of astaxanthin with different configurations is poor, and the number of peaks is small. The separation effect is inferior to that of the supercritical fluid chromatography.
Comparative example 2
The chromatograph selected is a high performance liquid chromatograph of Jiangsu Hanbang science and technology Limited, the specification of the chromatographic column is 10 multiplied by 250mm, the flow rate of a mobile phase system is 2mL/min, the concentration of the haematococcus pluvialis extract is 200mg/mL (the mass content of astaxanthin in the haematococcus pluvialis extract is 10%), the sample injection volume is 1 mu L, and the detection wavelength is 472 nm.
Table 8 shows the conditions for separating astaxanthin from the Haematococcus pluvialis extract solution in comparative example 2 by high performance liquid chromatography.
TABLE 8 HPLC SEPARATION CONDITIONS OF COMPARATIVE EXAMPLE 2
Figure BDA0003423605510000122
Figure BDA0003423605510000131
The specific steps corresponding to comparative example 2 are: a haematococcus pluvialis extract containing 10% by mass of astaxanthin is dissolved in ethyl acetate, ultrasonic assisted dissolution is carried out, a solution with the concentration of 200mg/mL is prepared, a Nucifera C30C 10 mu m 10 multiplied by 250mm is selected as a chromatographic column on a high performance liquid chromatograph of Jiangsu Hanbang science and technology Limited, and the volume ratio, the total flow rate and the temperature of the chromatographic column in a mobile phase A and B are controlled according to a table 8. The separation effect of the ethyl acetate solution of haematococcus pluvialis extract is shown in figure 8. As can be seen from fig. 8: the separation effect of astaxanthin with different configurations in high performance liquid chromatography by using a Nucifera C30C 10 multiplied by 250mm semi-preparative chromatographic column is poor, the number of peaks is small, and a mobile phase system is complex, so that the preparation of a large amount of astaxanthin is inconvenient.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A method for separating haematococcus pluvialis extract by supercritical fluid chromatography is characterized by comprising the following steps:
dissolving Haematococcus pluvialis extract in a low-polarity solvent to obtain a Haematococcus pluvialis extract solution;
performing supercritical fluid chromatographic separation on the haematococcus pluvialis extract solution;
the parameters of the supercritical fluid chromatographic separation include:
the chromatographic column comprises a modified silica gel chromatographic column;
the filler of the modified silica gel chromatographic column is silica gel with the surface bonded with pyridine, amino, phenyl, cyano, diol group, C18C, C18P or C18H;
the mobile phase system comprises a mobile phase A and a mobile phase B; the mobile phase A is supercritical carbon dioxide, and the mobile phase B is lower alcohol;
in the process of gradient elution, the volume ratio of the mobile phase A to the mobile phase B is 99-50: 1 to 50.
2. The method of claim 1, wherein the low polarity solvent comprises ethyl acetate or dichloromethane.
3. The method according to claim 1 or 2, wherein the concentration of the haematococcus pluvialis extract solution is 30 to 200 mg/mL.
4. The method of claim 1, wherein the lower alcohol comprises methanol or ethanol.
5. The method of claim 1, wherein the parameters of supercritical fluid chromatography further comprise: the detector is a PAD detector; the detection wavelength of the PAD detector is 472 nm.
6. The method according to claim 1, wherein when the packing material of the modified silica gel chromatographic column is silica gel with C18C bonded on the surface, the procedure of gradient elution is as follows:
0 min: the volume fraction of the supercritical carbon dioxide is 90 percent, and the volume fraction of the ethanol is 10 percent;
0-15 min: the volume fraction of the supercritical carbon dioxide is maintained to be 90 percent;
15-30 min; the volume fraction of the supercritical carbon dioxide is changed from 90% to 50% at a constant speed.
7. The method according to claim 1, wherein when the packing material of the modified silica gel chromatographic column is silica gel with C18C bonded on the surface, the procedure of gradient elution is as follows:
0 min: the volume fraction of the supercritical carbon dioxide is 99 percent, and the volume fraction of the ethanol is 1 percent;
0-50 min: the volume fraction of the supercritical carbon dioxide is maintained at 99 percent;
50-51 min: the volume fraction of the supercritical carbon dioxide is changed from 90% to 50% at a constant speed;
51-70 min: the volume fraction of supercritical carbon dioxide was maintained at 50%.
8. The method according to claim 1, wherein when the packing material of the modified silica gel chromatographic column is silica gel with C18P bonded on the surface, the procedure of gradient elution is as follows:
0 min: the volume fraction of the supercritical carbon dioxide is 97 percent, and the volume fraction of the ethanol is 3 percent;
0-30 min: the volume fraction of supercritical carbon dioxide was maintained at 97%.
9. The method according to claim 1, wherein when the packing material of the modified silica gel chromatographic column is silica gel with amino groups bonded on the surface, the procedure of gradient elution is as follows:
0 min: the volume fraction of the supercritical carbon dioxide is 92 percent, and the volume fraction of the ethanol is 8 percent;
0-20 min: the volume fraction of the supercritical carbon dioxide is maintained at 92%;
20-25 min; the volume fraction of the supercritical carbon dioxide is changed from 92% to 50% at a constant speed.
10. The method of claim 9, wherein the size of the chromatography column comprises 4.6 x 250mm, 10 x 250mm, 20 x 250mm, or 50 x 250 mm.
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