CN111330556A - Preparation method of chromatographic separation medium - Google Patents
Preparation method of chromatographic separation medium Download PDFInfo
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- CN111330556A CN111330556A CN202010153915.1A CN202010153915A CN111330556A CN 111330556 A CN111330556 A CN 111330556A CN 202010153915 A CN202010153915 A CN 202010153915A CN 111330556 A CN111330556 A CN 111330556A
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- chromatographic separation
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- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
Abstract
The invention relates to a preparation method of a chromatographic separation medium, which is characterized by comprising the following specific steps: (1) adding a certain amount of porous amino silicon balls into an organic solvent, stirring to wet the porous amino silicon balls, and uniformly dispersing; (2) adding 8-carboxyflavone into the organic solvent in which the porous amino silicon spheres are suspended, stirring and uniformly mixing; (3) adding a catalyst, heating for reaction for a period of time, and filtering; (4) washing with organic solvent for several times, heating and drying; obtaining chromatographic separation medium. The chromatographic medium bonded with the flavone group has the double effects of adsorbing phenolic hydroxyl groups by amide bonds, benzene rings by the flavone group and the like, so that the separation selectivity of the chromatographic medium is higher due to the common filler, especially the preparation load capacity, and the purification efficiency of the anthocyanins can be obviously improved.
Description
Technical Field
The invention relates to the technical field of chromatography, in particular to a preparation method of a chromatographic separation medium.
Background
Anthocyanins are natural pigments which are widely and purely found in plants in nature and are closely related to the colors of fruits and vegetables. The anthocyanidin can be combined with protein to prevent peroxidation, and has effects of resisting mutation, preventing cardiovascular disease and cerebrovascular disease, protecting liver, enhancing immune system to resist cancer, and improving vision. The cranberry has wide planting area and high yield, and anthocyanin in the cranberry can effectively inhibit helicobacter pylori, resist bacterial gastric ulcer, reduce incidence rate of gastric cancer and cardiovascular diseases, prevent senile dementia and maintain young and healthy skin.
At present, most of pigments used in the food industry are synthetic pigments, almost all of which are toxic and harm human health after long-term use, so that the extraction and purification of natural pigments in plants attract attention in the scientific and technological field. The separation and purification process of anthocyanidin generally comprises the following methods:
the liquid-liquid extraction method adopts an organic solvent to purify by a liquid-phase extraction method, the dosage of the solvent is large, the separation effect is poor, the quality of the obtained product is low, and the residues of toxic substances in the product are easy to cause. Patent CN 106243075A discloses a separation method of blueberry anthocyanin, which comprises the steps of crushing blueberry fruits, extracting with acidified ethanol, evaporating for drying, and extracting the dried product with n-hexane to obtain the blueberry anthocyanin.
The purity of the anthocyanin extracted and purified by the column chromatography can reach more than 90 percent, but the yield is very low. Patent CN200710037281.8 discloses a method for extracting procyanidin from cranberry, which comprises the steps of treating raw materials to obtain an extracting solution, degreasing, purifying by column chromatography, wherein organic solvents are easy to remain, and the solubility is poor, so that the method has limitations.
The macroporous resin method separation process is to separate the crude anthocyanin by a macroporous resin chromatographic column, then elute by an organic solvent, concentrate and dry to obtain a product, and the method has higher relation with the selectivity of the macroporous resin and can not stably control the purity. The patent CN 102336794A discloses a method for extracting and separating malvidin-3-0-glucoside from cranberry, which comprises the steps of water extraction, macroporous resin adsorption, ethanol desorption, polyamide adsorption, ethanol gradient elution, concentration and drying to obtain the product. Patent CN 105294633a discloses an industrialized method for preparing cranberry anthocyanin from cranberries, which comprises the steps of extracting with an acid aqueous solution, removing impurities by flocculation, purifying by macroporous resin adsorption, concentrating under reduced pressure, and spray drying to obtain a product.
The microbiological method is to decompose the cell wall of the plant by the degradation of the microorganism, then to extract with water, to purify by macroporous resin, to concentrate, to dry to obtain the product. Patent CN 106632204a discloses a method for extracting and purifying anthocyanins from cranberries, and the anthocyanins obtained by the method are low in yield and contain a certain proportion of anthocyanins.
The supercritical fluid extraction technology adopts pure carbon dioxide as a fluid solvent, is safe and nontoxic, and the carbon dioxide has special permeability and dissolving capacity to materials in a supercritical state. The density of the supercritical carbon dioxide is very sensitive to temperature and pressure changes, and the supercritical fluid is changed into common gas and separated from extracted substances by controlling the density, the pressure and the temperature of the carbon dioxide, so that the purposes of separation and purification are achieved. Patent CN 109761946A discloses a method for extracting and separating cranberry anthocyanin, which uses ethanol extraction combined with supercritical fluid separation to purify carbon dioxide, and the purity of the cranberry anthocyanin is up to 99% after twice crystallization.
The conventional purification method has the disadvantages of complicated process, complex operation, high cost and low yield. And the anthocyanin is easily degraded due to high extraction temperature. Chromatography is a mode with high utilization rate in the separation and purification process of natural products, but the chromatography is high in cost, the purity of the products is greatly influenced by packing, and the yield efficiency in preparation is low. In order to solve the problems, the patent provides a preparation method of a special chromatographic column filler for separating and purifying anthocyanin, flavone with carboxyl is bonded to a silicon sphere, amido bond and benzene ring respectively generate adsorption to phenolic hydroxyl and benzene ring on the anthocyanin, retention of the anthocyanin on a chromatographic column is enhanced, product purity is high, the special chromatographic column filler can be used for preparing chromatography, and industrial production requirements are met.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a preparation method of a chromatographic separation medium.
The purpose of the invention is realized by the following technical scheme:
a method for preparing chromatographic separation medium comprises bonding flavone containing carboxyl onto silicon ball to obtain filler containing two effective groups such as flavone and amido bond; the method comprises the following specific steps:
(1) adding the porous amino silicon balls into an organic solvent, stirring to wet the porous amino silicon balls, and uniformly dispersing;
the particle size of the porous amino silicon ball is 20-200 microns.
The organic solvent is toluene or carbon tetrachloride, and toluene is preferred.
The mass ratio of the porous amino silicon spheres to the organic solvent is 2: 5-2: 7.
(2) Adding 8-carboxyflavone into the organic solvent in which the porous amino silicon spheres are suspended, stirring and uniformly mixing;
the dosage of the 8-carboxyflavone is 30-70% of the mass of the porous amino silicon ball.
(3) Adding a catalyst, heating for reaction for a period of time, and filtering;
the catalyst is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide or N-hydroxysuccinimide, preferably N-hydroxysuccinimide.
The dosage of the catalyst is 0.1-5% of the mass of the 8-carboxyflavone.
The heating temperature is 60-80 ℃, the heating time is 18-24 hours, and the heating time is preferably 20 hours, wherein 75 ℃ is preferred.
(4) Washing with organic solvent for several times, heating and drying; obtaining chromatographic separation medium.
The organic solvent used for washing is toluene or toluene which is preferably selected from carbon tetrachloride, and the drying temperature is 60-80 ℃, and is preferably 75 ℃.
Compared with the prior art, the invention has the following positive effects:
the bonded chromatographic separation medium disclosed by the invention has the advantages that 1) the amido bond and the group contained in the medium have double effects on anthocyanin, the anthocyanin can be selectively purified, and the medium is a special filler for purifying various anthocyanidins; 2) the preparation method of the medium is simple and low in cost; 3) by adopting the chromatographic technology, the high-purity anthocyanin can be obtained, the purification yield is high, and the industrialization is easy to realize.
Drawings
FIG. 1 shows a conventional stationary phase bonding group structure;
FIG. 2 is a chemical synthesis scheme of the present invention;
FIG. 3 is a liquid chromatography analysis chart of a purchased anthocyanin standard;
FIG. 4 is a liquid chromatography analysis of crude cranberry anthocyanins from example 1;
FIG. 5 is a liquid chromatography analysis of crystals of crude cranberry anthocyanin after purification in example 1;
FIG. 6 is a liquid chromatography analysis of crude cranberry anthocyanins from example 2;
fig. 7 is a liquid chromatography analysis chart of crystals after purification of crude cranberry anthocyanin in example 2.
Detailed Description
The following provides a specific embodiment of a method of preparing a chromatographic separation medium of the present invention.
Example 1
Cranberry, also known as cranberry, is commonly known as evergreen shrub, a subgenus of cranberry belonging to the family Ericaceae, and grows in cool, acidic peat soil in the northern hemisphere. One of three traditional fruits in North America, cranberry is a high-activity functional small berry, and the fruit is rich in various active substances such as organic acid, vitamin C, flavonoid compounds, anthocyanin, procyanidine and the like.
This example shows the preparation method of the present chromatographic separation medium by taking cranberry anthocyanidin as an example. The flavone containing carboxyl is bonded on the surface of the amino porous silicon sphere, so that the surface of the silicon sphere is more effective in separating the cranberry anthocyanin. The method comprises the following specific steps:
(1) adding 20g of porous amino silicon balls with the particle size of 50 mu m into 50g of toluene solution, and uniformly stirring to fully wet the porous amino silicon balls;
(2) adding 9g of 8-carboxyflavone into a toluene solution in which porous amino silicon spheres are suspended, stirring and uniformly mixing;
(3) then adding 0.1g N-hydroxysuccinimide, reacting for 20h at 75 ℃, and filtering to obtain bonded silicon spheres;
(4) and washing the bonded silicon spheres with toluene for 3 times, drying at 75 ℃ to obtain a chromatographic separation medium, and filling into a chromatographic column.
Column 15 × 310mm, 50 μm,
wavelength: 210nm
Mobile phase: methanol-water
Flow rate: 10.6mL/min
10g of crude cranberry anthocyanin extracted by solvent method was analyzed by C18 reverse phase chromatography, as shown in FIG. 4. The isolation and purification of cranberry anthocyanins was carried out using a packed preparative column to give 1.15g of crystals which were analysed using a C18 reverse phase column with a purity of up to 95.04%, see FIG. 5.
Example 2
The method comprises the following steps of changing the mixture ratio of raw materials and a plurality of reagents, and preparing the cranberry anthocyanin separation special medium, wherein the method comprises the following specific steps:
(1)20g of porous amino silicon balls with the particle size of 200 mu m are added into 70g of toluene solution and stirred uniformly to be fully wetted;
(2) adding 10g of 8-carboxyflavone into a toluene solution in which porous amino silicon spheres are suspended, stirring and uniformly mixing;
(3) then 0.2g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is added, the reaction is carried out for 20 hours at the temperature of 75 ℃, and the bonded silica spheres are obtained after filtration;
(4) and washing the bonded silicon spheres with toluene for 3 times, drying at 75 ℃ to obtain a chromatographic separation medium, and filling into a chromatographic column.
Column 15 × 310mm, 200 μm,
wavelength: 210nm
Mobile phase: methanol-water
Flow rate: 10.6mL/min
10g of crude cranberry anthocyanin extracted by solvent method was analyzed by C18 reverse phase chromatography, as shown in FIG. 6. The isolation and purification of cranberry anthocyanins was carried out using a packed preparative column to give 4.2g of crystals which were analysed using a C18 reverse phase column with a purity as high as 95.78%, see FIG. 7.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and decorations can be made without departing from the concept of the present invention, and these modifications and decorations should also be regarded as being within the protection scope of the present invention.
Claims (9)
1. A method for preparing chromatographic separation medium is characterized in that flavone containing carboxyl is bonded to silicon spheres, and the obtained filler contains two effective groups of flavone, amido bond and the like; the method comprises the following specific steps:
(1) adding the porous amino silicon balls into an organic solvent, stirring to wet the porous amino silicon balls, and uniformly dispersing;
(2) adding 8-carboxyflavone into the organic solvent in which the porous amino silicon spheres are suspended, stirring and uniformly mixing;
(3) adding a catalyst, heating for reaction, and filtering;
(4) washing with organic solvent for several times, heating and drying; obtaining chromatographic separation medium.
2. A method of preparing a chromatographic separation medium according to claim 1 wherein in step (1) the porous aminosilicone spheres have a particle size of from 20 to 200 microns.
3. A process for the preparation of a chromatographic medium according to claim 1 wherein in step (1) the organic solvent is toluene or carbon tetrachloride, preferably toluene.
4. The method for preparing a chromatographic separation medium according to claim 1, wherein in the step (1), the mass ratio of the porous amino silicon spheres to the organic solvent is 2:5 to 2: 7.
5. The method for preparing a chromatographic separation medium according to claim 1, wherein in the step (2), the amount of the 8-carboxyflavone is 30-70% by mass of the porous aminosilicone spheres.
6. A process for the preparation of a chromatographic separation medium according to claim 1 wherein in step (2) the catalyst is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide or N-hydroxysuccinimide, preferably N-hydroxysuccinimide.
7. The method for preparing a chromatographic separation medium according to claim 1, wherein in the step (3), the amount of the catalyst is 0.1-5% by mass of the 8-carboxyflavone.
8. A process for the preparation of a chromatographic separation medium according to claim 1 wherein in step (3) the heating is carried out at a temperature of 60 to 80 ℃, preferably 75 ℃ for a time of 18 to 24 hours, preferably 20 hours.
9. The method of claim 1, wherein in step (4), the organic solvent used for washing is toluene or carbon tetrachloride, preferably toluene, and the drying temperature is 60 to 80 ℃, preferably 75 ℃.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114602438A (en) * | 2020-12-08 | 2022-06-10 | 苏州汇通色谱分离纯化有限公司 | Preparation method of special chromatographic medium for separating hypocannabidiol |
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Effective date of registration: 20220923 Address after: No. 120, Building 3, No. 926, Changhong East Street, Fuxi Street, Deqing County, Huzhou City, Zhejiang Province, 313299 (Moganshan National High-tech Zone) Applicant after: ZHEJIANG RUNHE BIOLOGY TECHNOLOGY CO.,LTD. Address before: Room 3-1101, No. 926, Changhong East Street, Fuxi Street, Deqing County, Huzhou City, Zhejiang Province 323200 Applicant before: Zhejiang peptide Biotechnology Development Co.,Ltd. |
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