CN103588741A - Method for separating and purifying single high-purity flavonoid compound by using SFC (supercritical fluid chromatography) - Google Patents
Method for separating and purifying single high-purity flavonoid compound by using SFC (supercritical fluid chromatography) Download PDFInfo
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- CN103588741A CN103588741A CN201310568685.5A CN201310568685A CN103588741A CN 103588741 A CN103588741 A CN 103588741A CN 201310568685 A CN201310568685 A CN 201310568685A CN 103588741 A CN103588741 A CN 103588741A
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- flavonoid compound
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
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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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/40—Separation, e.g. from natural material; Purification
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- 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
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a method for separating and purifying a single high-purity flavonoid compound by using SFC (supercritical fluid chromatography). A propolis sample containing the flavonoid compound is injected into a mobile phase, so that the mobile phase containing the sample passes through a chromatographic column which is filled with a flavonoid compound separating agent, and the single high-purity flavonoid compound can be obtained, wherein the mobile phase is a supercritical CO2 fluid added with a modifier. According to the method for separating and purifying the single high-purity flavonoid compound by using the SFC, the chromatographic column which is filled with the flavonoid compound separating agent is used, the supercritical CO2 fluid added with the modifier is taken as the mobile phase, chromatogram separation purification is performed, and the single high-purity flavonoid compound can be obtained on the basis that technical parameters such as supercritical CO2 fluid temperature, the pressure, modifier types and the like are selected.
Description
Technical field
The present invention relates to a kind of method of separation and purification of flavonoid compound.
Background technology
Supercritical fluid chromatography (supercritical fluid chromatography, be called for short SFC) is to take the chromatography that supercutical fluid is moving phase.Supercutical fluid is a kind of state of material when higher than emergent pressure and critical temperature, it has some character of gas and liquid, there is the low viscosity of gas, the high-density of liquid and the features such as spread coefficient between between gas, liquid, SFC is supplementing of GC and LC, SFC can solve a difficult problem for gas-liquid chromatograph separation, it can segregation gas chromatography the non-volatile substance of difficult vaporization, there is the efficiency higher than high performance liquid chromatography simultaneously, analysis time is shorter.Especially its supercutical fluid used is generally carbonic acid gas, and with traditional liquid phase comparison, the Discarded organic solvent of generation is few, pollute little, so supercritical fluid chromatography technology has broad application prospects.
Propolis (propolis) is the resinous substance that honeybee gathers collagen plant plumule and innovation leaf bud, at the secretory product that mixes the cured gland of honeybee lingual gland, mixes a kind of gluey natural active matter.A large amount of researchs show, that propolis has is antibiotic, anti-inflammatory, inhibition is viral, anticancer and increase physiology and the pharmacological functions such as body's immunity, and in propolis, contained flavonoid, terpenes, polysaccharide, enzyme, organic acid etc. have again unique effect to cancer patients.Various countries scientist studies have shown that total Flavones From Propolis has obvious restraining effect and antitumous effect to cancer cells core.
Research and development PTS need to obtain single high purity flavonoid compound, the method of supercritical carbon dioxide extraction only can obtain the flavonoid compound mixing, and traditional high performance liquid chromatography (HPLC) method, though can obtain single high purity flavonoid compound, but the Discarded organic solvent producing is many, pollute greatly length consuming time.Therefore fast, efficiently, the single high purity flavonoid compound that obtains of economy, environmental protection is the important topic that urgent need will solve.
Summary of the invention
The technical problem to be solved in the present invention is to overcome existing defect, provide a kind of fast, the method for the single high purity flavonoid compound of supercritical fluid chromatography separation and purification of efficient, economy, environmental protection.
Object of the present invention is carried out specific implementation by the following technical programs:
The method of the single high purity flavonoid compound of a kind of supercritical fluid chromatography separation and purification, the propolis sample that contains flavonoid compound is injected to moving phase, make the moving phase that contains sample by having filled the chromatographic column of flavonoid compound separating agent, can obtain single high purity flavonoid compound, wherein: described moving phase is the supercritical CO that is added with properties-correcting agent
2fluid.
Further, this chromatographic separation process is a circulating enclosed system, comprises the steps:
1) preparation of sample
The propolis that contains flavonoid compound is dissolved in organic heavy solvent, obtains propolis sample;
2) preparation of moving phase
CO2 gas is made into supercritical CO
2fluid, adds after properties-correcting agent, forms moving phase;
3) separation
By propolis sample with the moving phase that is added with properties-correcting agent by being filled with the chromatographic column (existing conventional chromatogram post is commonly used and had C-18 post, phenyl post, itrile group post) of flavonoid compound separating agent, obtain respectively the flavonoid compound that high purity is single;
4) reclaim
The CO producing in chromatographic separation process
2waste gas, continues to be made into supercritical CO after purifying
2moving phase; The waste liquid producing in chromatographic separation process, purifies the organic solvent reclaiming as propolis.
Preferably,
Described organic heavy solvent adopts methyl alcohol or cyclohexane;
Described supercritical CO
2the pressure of fluid is that 20-40MPa, temperature are 40 ℃.
Described properties-correcting agent is supercritical CO
2the polar liquid material of 5wt% to 30wt% of moving phase total amount.
More excellent,
Described polar liquid material is to be selected from water, acetonitrile, methyl alcohol, ethanol, Virahol.
Best, described properties-correcting agent is water, accounts for 10% of moving phase total amount.
Beneficial effect of the present invention:
The method of the single high purity flavonoid compound of supercritical fluid chromatography separation and purification provided by the present invention, utilizes the chromatographic column of having filled flavonoid compound separating agent, to be added with the supercritical CO of properties-correcting agent
2as moving phase, carry out the separation and purification of chromatogram, by screening supercritical CO
2on the basis of the technical parameters such as fluid temperature (F.T.), pressure, properties-correcting agent kind, can obtain single high purity flavonoid compound.Adding of properties-correcting agent, improve flavonoid compound separation efficiency, with traditional separation purification method comparison; the Discarded organic solvent that the method for the present patent application protection overcomes existing separation purification method is many; pollute greatly, the defect of length consuming time, has fast, the characteristic of efficient, economy, environmental protection.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for specification sheets, for explaining the present invention, is not construed as limiting the invention together with embodiments of the present invention.In the accompanying drawings:
Fig. 1 is process flow sheet of the present invention;
Fig. 2 is the uv-absorbing spectrogram of the propolis sample that contains flavonoid compound;
Fig. 3 is the infrared spectrum of the isolated kaempferide of technique of the present invention;
Fig. 4 is the infrared spectrum of the isolated ampelopsin of technique of the present invention;
Fig. 5 is the infrared spectrum of the isolated Quercetin of technique of the present invention.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein, only for description and interpretation the present invention, is not intended to limit the present invention.
The preparation of sample:
By the propolis 10g of the flavonoid compound containing, be dissolved in the methanol solution of 50ml, its uv-absorbing spectrogram is referring to accompanying drawing 2, in the drawings, ordinate zou represents the intensity of uv-absorbing, and X-coordinate represents the time, and an absorption peak represents a composition, also with regard to each constituents in this mixture in the intensity of each different time uv-absorbing, i.e. concentration.As can be seen here, various flavonoid compounds shared ratio in propolis.
Chromatographic column is selected c-18 post.
embodiment 1:
Get the feeding unit that 5ml sample injects supercritical fluid chromatography purifies and separates system, mix by divider and then flow into and filled flavonoid compound separating agent separation chromatography post with the supercritical carbon dioxide fluid that adds properties-correcting agent water, detection through isolate detector, obtain that respectively cancer cells core is had to the single flavonoid compound of obvious inhibiting high purity, accompanying drawing 3, 4, 5 have enumerated respectively three flavonoid compounds wherein: kaempferide, ampelopsin, Quercetin, as can be seen from Figure, isolated material is single compound, without other impurity.Supercritical co pressure 25mpa in this example, controls 40 ℃ of insulation can temperature, at CO
2fluid is by divider time, and constantly incorporating modified dose of water, accounts for CO
210% of fluid total volume.
embodiment 2:
Get the feeding unit that 5ml sample injects supercritical fluid chromatography purifies and separates system, mix by divider and then flow into and filled flavonoid compound separating agent separation chromatography post with the supercritical carbon dioxide fluid that adds properties-correcting agent acetonitrile, detection through isolate detector, obtains that equally respectively cancer cells core is had to the single flavonoid compound of obvious inhibiting high purity.Supercritical co pressure 20mpa in this example, controls 40 ℃ of insulation can temperature, at CO
2fluid is by divider time, and constantly incorporating modified dose of acetonitrile, accounts for CO
25% of fluid total volume.
embodiment 3:
Get the feeding unit that 5ml sample injects supercritical fluid chromatography purifies and separates system, mix by divider and then flow into and filled flavonoid compound separating agent separation chromatography post with the supercritical carbon dioxide fluid that adds properties-correcting agent acetonitrile, detection through isolate detector, obtains that equally respectively cancer cells core is had to the single flavonoid compound of obvious inhibiting high purity.Supercritical co pressure 35mpa in this example, controls 40 ℃ of insulation can temperature, at CO
2fluid is by divider time, and constantly incorporating modified dose of acetonitrile, accounts for CO
230% of fluid total volume.
embodiment 4:
Get the feeding unit that 5ml sample injects supercritical fluid chromatography purifies and separates system, mix by divider and then flow into and filled flavonoid compound separating agent separation chromatography post with the supercritical carbon dioxide fluid that adds properties-correcting agent acetonitrile, detection through isolate detector, obtains that equally respectively cancer cells core is had to the single flavonoid compound of obvious inhibiting high purity.Supercritical co pressure 25mpa in this example, controls 40 ℃ of insulation can temperature, at CO
2fluid is by divider time, and constantly incorporating modified dose of acetonitrile, accounts for CO
220% of fluid total volume.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although the present invention is had been described in detail with reference to previous embodiment, for a person skilled in the art, its technical scheme that still can record aforementioned each embodiment is modified, or part technical characterictic is wherein equal to replacement.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (7)
1. the method for the single high purity flavonoid compound of supercritical fluid chromatography separation and purification, it is characterized in that: the propolis sample that contains flavonoid compound is injected to moving phase, make the moving phase that contains sample by having filled the chromatographic column of flavonoid compound separating agent, can obtain single high purity flavonoid compound, wherein: described moving phase is the supercritical CO that is added with properties-correcting agent
2fluid.
2. the method for the single high purity flavonoid compound of supercritical fluid chromatography separation and purification according to claim 1, is characterized in that: this chromatographic separation process is a circulating enclosed system, comprises the steps:
1) preparation of sample
The propolis that contains flavonoid compound is dissolved in organic solvent, obtains propolis sample;
2) preparation of moving phase
By CO
2gas is made into supercritical CO
2fluid, adds after properties-correcting agent, forms moving phase;
3) separation
By propolis sample with the moving phase that is added with properties-correcting agent by being filled with the chromatographic column of flavonoid compound separating agent, obtain respectively the flavonoid compound that high purity is single;
4) reclaim
The CO producing in chromatographic separation process
2waste gas, continues to be made into supercritical CO after purifying
2moving phase; The waste liquid producing in chromatographic separation process, purifies the organic solvent reclaiming as propolis.
3. the method for the single high purity flavonoid compound of supercritical fluid chromatography separation and purification according to claim 2, is characterized in that: described organic solvent adopts methyl alcohol or hexanaphthene.
4. the method for the single high purity flavonoid compound of supercritical fluid chromatography separation and purification according to claim 1 and 2, is characterized in that: described supercritical CO
2the pressure of fluid is that 20-40MPa, temperature are 40 ℃.
5. the method for the single high purity flavonoid compound of supercritical fluid chromatography separation and purification according to claim 1 and 2, is characterized in that: described properties-correcting agent is supercritical CO
2the polar liquid material of 5wt% to 30wt% of moving phase total amount.
6. the method for the single high purity flavonoid compound of supercritical fluid chromatography separation and purification according to claim 5, is characterized in that: described polar liquid material is to be selected from water, acetonitrile, methyl alcohol, ethanol, Virahol.
7. the method for the single high purity flavonoid compound of supercritical fluid chromatography separation and purification according to claim 6, is characterized in that: described properties-correcting agent is water, accounts for 10% of moving phase total amount.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104971516A (en) * | 2014-04-10 | 2015-10-14 | 信越化学工业株式会社 | METHOD FOR PREPARING A COMPOSITION COMPRISING PERFLUOROPOLYETHER HAVING A HYDROXYL GROUP AT ONE TERMINAL and method for increasing the content ratio of the compound |
| CN106496023A (en) * | 2015-09-08 | 2017-03-15 | 庄明熙 | Method for preparing, separating and purifying artemisinin C active ingredient in propolis |
| CN107216338A (en) * | 2016-03-21 | 2017-09-29 | 江苏汉邦科技有限公司 | A kind of method for isolating and purifying moxidectin |
Citations (4)
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|---|---|---|---|---|
| JPH06276966A (en) * | 1993-03-30 | 1994-10-04 | Kumamoto Pref Gov | Processing of propolis |
| CN1493304A (en) * | 2003-07-25 | 2004-05-05 | 吴晓闻 | Method of extracting mixed propolis using supercritical fluid separation |
| CN1569040A (en) * | 2004-04-30 | 2005-01-26 | 无锡尚宝生物科技有限公司 | Hypercritical soluble propolis prescription and its preparing method |
| CN102218082A (en) * | 2010-04-13 | 2011-10-19 | 江山福赐德蜂业科技开发有限公司 | Method for extracting and purifying natural-state yellow bee propolis |
-
2013
- 2013-11-15 CN CN201310568685.5A patent/CN103588741A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06276966A (en) * | 1993-03-30 | 1994-10-04 | Kumamoto Pref Gov | Processing of propolis |
| CN1493304A (en) * | 2003-07-25 | 2004-05-05 | 吴晓闻 | Method of extracting mixed propolis using supercritical fluid separation |
| CN1569040A (en) * | 2004-04-30 | 2005-01-26 | 无锡尚宝生物科技有限公司 | Hypercritical soluble propolis prescription and its preparing method |
| CN102218082A (en) * | 2010-04-13 | 2011-10-19 | 江山福赐德蜂业科技开发有限公司 | Method for extracting and purifying natural-state yellow bee propolis |
Non-Patent Citations (2)
| Title |
|---|
| 刘志敏等: "黄酮类化合物的超临界流体色谱分离", 《分析化学》, vol. 25, no. 3, 31 March 1997 (1997-03-31) * |
| 孙晓莉等: "《色谱分析》", 31 October 2012, article "超临界流体色谱法", pages: 185 - 2-3 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104971516A (en) * | 2014-04-10 | 2015-10-14 | 信越化学工业株式会社 | METHOD FOR PREPARING A COMPOSITION COMPRISING PERFLUOROPOLYETHER HAVING A HYDROXYL GROUP AT ONE TERMINAL and method for increasing the content ratio of the compound |
| CN104971516B (en) * | 2014-04-10 | 2018-07-06 | 信越化学工业株式会社 | The method of the manufacturing method of the composition of per-fluoro polyether compound containing single end hydroxyl and the containing ratio of the raising compound |
| CN106496023A (en) * | 2015-09-08 | 2017-03-15 | 庄明熙 | Method for preparing, separating and purifying artemisinin C active ingredient in propolis |
| CN107216338A (en) * | 2016-03-21 | 2017-09-29 | 江苏汉邦科技有限公司 | A kind of method for isolating and purifying moxidectin |
| CN107216338B (en) * | 2016-03-21 | 2019-04-02 | 江苏汉邦科技有限公司 | A method of isolating and purifying moxidectin |
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Application publication date: 20140219 |