CN114605488A - Method for purifying glycyrrhizic acid by using preparative chromatography technology - Google Patents
Method for purifying glycyrrhizic acid by using preparative chromatography technology Download PDFInfo
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- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 title claims abstract description 53
- LPLVUJXQOOQHMX-UHFFFAOYSA-N glycyrrhetinic acid glycoside Natural products C1CC(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2=O)C(O)=O)C)(C)CC2)(C)C2C(C)(C)C1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O LPLVUJXQOOQHMX-UHFFFAOYSA-N 0.000 title claims abstract description 43
- 229960004949 glycyrrhizic acid Drugs 0.000 title claims abstract description 43
- 235000019410 glycyrrhizin Nutrition 0.000 title claims abstract description 43
- UYRUBYNTXSDKQT-UHFFFAOYSA-N glycyrrhizic acid Natural products CC1(C)C(CCC2(C)C1CCC3(C)C2C(=O)C=C4C5CC(C)(CCC5(C)CCC34C)C(=O)O)OC6OC(C(O)C(O)C6OC7OC(O)C(O)C(O)C7C(=O)O)C(=O)O UYRUBYNTXSDKQT-UHFFFAOYSA-N 0.000 title claims abstract description 42
- VTAJIXDZFCRWBR-UHFFFAOYSA-N Licoricesaponin B2 Natural products C1C(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2)C(O)=O)C)(C)CC2)(C)C2C(C)(C)CC1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O VTAJIXDZFCRWBR-UHFFFAOYSA-N 0.000 title claims abstract description 40
- 239000001685 glycyrrhizic acid Substances 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000004237 preparative chromatography Methods 0.000 title claims description 9
- 238000005516 engineering process Methods 0.000 title description 3
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- 239000000945 filler Substances 0.000 claims abstract description 14
- LPLVUJXQOOQHMX-IOHDZAKGSA-N (2s,3s,4s,5r,6r)-6-[(2s,3r,4s,5s,6s)-2-[[(3s,4ar,6ar,6bs,8as,11s,12as,14ar,14bs)-11-carboxy-4,4,6a,6b,8a,11,14b-heptamethyl-14-oxo-2,3,4a,5,6,7,8,9,10,12,12a,14a-dodecahydro-1h-picen-3-yl]oxy]-6-carboxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-c Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-IOHDZAKGSA-N 0.000 claims abstract description 12
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- 238000011068 loading method Methods 0.000 claims description 13
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- 235000006200 Glycyrrhiza glabra Nutrition 0.000 claims description 8
- 244000303040 Glycyrrhiza glabra Species 0.000 claims description 8
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 8
- 235000011477 liquorice Nutrition 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
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- 238000013375 chromatographic separation Methods 0.000 claims description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 6
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- -1 diamino Chemical group 0.000 claims description 4
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- 238000001179 sorption measurement Methods 0.000 claims description 4
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 3
- 235000011054 acetic acid Nutrition 0.000 claims description 3
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- 125000005647 linker group Chemical group 0.000 claims description 3
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical group [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- 229910052801 chlorine Inorganic materials 0.000 claims description 2
- 125000001309 chloro group Chemical group Cl* 0.000 claims description 2
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 2
- KSFBTBXTZDJOHO-UHFFFAOYSA-N diaminosilicon Chemical compound N[Si]N KSFBTBXTZDJOHO-UHFFFAOYSA-N 0.000 claims description 2
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- 235000006408 oxalic acid Nutrition 0.000 claims description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 2
- PARWUHTVGZSQPD-UHFFFAOYSA-N phenylsilane Chemical compound [SiH3]C1=CC=CC=C1 PARWUHTVGZSQPD-UHFFFAOYSA-N 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 238000007789 sealing Methods 0.000 claims description 2
- FZHAPNGMFPVSLP-UHFFFAOYSA-N silanamine Chemical compound [SiH3]N FZHAPNGMFPVSLP-UHFFFAOYSA-N 0.000 claims description 2
- 229910000077 silane Inorganic materials 0.000 claims description 2
- 229910052710 silicon Inorganic materials 0.000 claims description 2
- 239000010703 silicon Substances 0.000 claims description 2
- LCHWKMAWSZDQRD-UHFFFAOYSA-N silylformonitrile Chemical compound [SiH3]C#N LCHWKMAWSZDQRD-UHFFFAOYSA-N 0.000 claims description 2
- MVMVARHTRUEHCL-UHFFFAOYSA-N [SiH4].[Br] Chemical compound [SiH4].[Br] MVMVARHTRUEHCL-UHFFFAOYSA-N 0.000 claims 1
- HICCMIMHFYBSJX-UHFFFAOYSA-N [SiH4].[Cl] Chemical compound [SiH4].[Cl] HICCMIMHFYBSJX-UHFFFAOYSA-N 0.000 claims 1
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- 239000000523 sample Substances 0.000 description 12
- 239000003814 drug Substances 0.000 description 6
- ILRKKHJEINIICQ-OOFFSTKBSA-N Monoammonium glycyrrhizinate Chemical compound N.O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@H]1CC[C@]2(C)[C@H]3C(=O)C=C4[C@@H]5C[C@](C)(CC[C@@]5(CC[C@@]4(C)[C@]3(C)CC[C@H]2C1(C)C)C)C(O)=O)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O ILRKKHJEINIICQ-OOFFSTKBSA-N 0.000 description 5
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- 241000196324 Embryophyta Species 0.000 description 2
- 241000220485 Fabaceae Species 0.000 description 2
- 239000004378 Glycyrrhizin Substances 0.000 description 2
- 238000011097 chromatography purification Methods 0.000 description 2
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- MPDGHEJMBKOTSU-NFYGSHHYSA-N (2s,4as,6ar,6as,6br,10s,12as,14bs)-10-hydroxy-2,4a,6a,6b,9,9,12a-heptamethyl-13-oxo-3,4,5,6,6a,7,8,8a,10,11,12,14b-dodecahydro-1h-picene-2-carboxylic acid Chemical compound C([C@@H]1C2=CC(=O)[C@H]34)[C@@](C)(C(O)=O)CC[C@]1(C)CC[C@@]2(C)[C@]4(C)CCC1[C@]3(C)CC[C@H](O)C1(C)C MPDGHEJMBKOTSU-NFYGSHHYSA-N 0.000 description 1
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 description 1
- MPDGHEJMBKOTSU-YKLVYJNSSA-N 18beta-glycyrrhetic acid Chemical compound C([C@H]1C2=CC(=O)[C@H]34)[C@@](C)(C(O)=O)CC[C@]1(C)CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@H](O)C1(C)C MPDGHEJMBKOTSU-YKLVYJNSSA-N 0.000 description 1
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- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
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- 206010011878 Deafness Diseases 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 208000005392 Spasm Diseases 0.000 description 1
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- VQPFDLRNOCQMSN-UHFFFAOYSA-N bromosilane Chemical compound Br[SiH3] VQPFDLRNOCQMSN-UHFFFAOYSA-N 0.000 description 1
- KOPOQZFJUQMUML-UHFFFAOYSA-N chlorosilane Chemical compound Cl[SiH3] KOPOQZFJUQMUML-UHFFFAOYSA-N 0.000 description 1
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- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
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- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- ZDYUUBIMAGBMPY-UHFFFAOYSA-N oxalic acid;hydrate Chemical compound O.OC(=O)C(O)=O ZDYUUBIMAGBMPY-UHFFFAOYSA-N 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
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- OVARTBFNCCXQKS-UHFFFAOYSA-N propan-2-one;hydrate Chemical compound O.CC(C)=O OVARTBFNCCXQKS-UHFFFAOYSA-N 0.000 description 1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
-
- 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
Abstract
The invention provides a method for separating and purifying glycyrrhizic acid by using a preparation chromatogram, which takes a licorice extract as a raw material, realizes the purification of glycyrrhizic acid by using a preparation chromatogram separation and purification means, adopting a silica gel filler modified by polar groups and using an organic solvent as an eluent, and obtains glycyrrhizic acid products with the content of more than 90 percent (related substance impurity A is less than 3 percent, isomer impurity 18 alpha glycyrrhizic acid is less than 5 percent). The method can effectively remove isomer impurities, controls the limited impurity content, has the characteristics of high recovery rate, high product content, low solvent consumption, good stability, simple process flow, simple and controllable operation and the like, and is suitable for separation and purification of production.
Description
Technical Field
The method relates to the technical field of separation and purification of medicinal plant components, in particular to a method for separating and purifying glycyrrhizic acid by utilizing a preparation chromatogram, wherein the content of a product is more than 90 percent (related substance impurity A is less than 3 percent, isomer impurity 18 alpha glycyrrhizic acid is less than 5 percent).
Background
The liquorice is a plant of liquorice of Leguminosae (Leguminosae) Papiliantae subfamily (Papiliantae Taub), is an important traditional Chinese medicine in China, has the effects of tonifying spleen and qi, eliminating phlegm and relieving cough, relieving spasm and pain, clearing heat and detoxicating, harmonizing the medicines and the like, and is a medicine and food. The main active ingredients of the glycyrrhizic acid are glycyrrhizic acid (also known as glycyrrhizin and glycyrrhizin), which is triterpenoid saponin extracted from dried roots and rhizomes of liquorice. In the pharmaceutical industry, glycyrrhizic acid is usually prepared into water-soluble glycyrrhetate, such as monoammonium glycyrrhizinate, which is used as a raw material for preparing the preparation. In 1992, monoammonium glycyrrhizinate is the first choice drug for treating chronic hepatitis by the national ministry of health. The main components of the monoammonium glycyrrhizinate are a pair of epimers, namely 18 alpha-glycyrrhizic acid (18 alpha-glycyrrhetic acid, 18 alpha-Gly) and 18 beta-glycyrrhizic acid (18 beta-glycyrrhetic acid, 18 beta-Gly), and the main components contain 2 related substances which are respectively named as impurity A and impurity B. The molecular formulas of the 18 alpha-Gly and the 18 beta-Gly are the same, the spatial configuration has slight difference, and the structure phase difference exists only in 18H, but the two have obvious difference in the aspects of pharmacology, pharmacodynamics, pharmacokinetic adverse reaction, clinical curative effect and the like.
Glycyrrhizic acid is white or light yellow crystal powder, has a melting point of 220 ℃, has special sweet taste and has a molecular formula: C42H62O16, molecular weight: 822.92, the structural formula is as follows:
the Chinese pharmacopoeia does not collect ammonium glycyrrhizinate raw material drugs or preparations, and has the current Chinese national drug standard (WS)1-XG-2002) only contains related preparations of glycyrrhizic acid, and only content detection is performed on the mixture of main components thereof, effective separation of 18 α -Gly and 18 β -Gly is not achieved, and no provision is made for related substance detection and limitation problems, resulting in loss of internal quality standards.
However, most foreign quality standards have specific requirements on related substances of the product, wherein in the quality standard of monoammonium glycyrrhizinate collected in European pharmacopoeia EP7.0 (EP7.0), 18 alpha-Gly is required to be not more than 10.0%, impurity A is required to be not more than 5.0%, any other impurity is required to be not more than 2.0%, and the sum of other total impurities is required to be not more than 7.0%. At present, some domestic production enterprises also have clear limitations on related substances of the product. Therefore, the method for separating the isomers of the glycyrrhizic acid main component and the related substances thereof is very important.
Due to similar structures, the separation of two isomers is very difficult, the common purification method cannot separate and has low yield, so that great waste is caused, and the main production method has the following defects:
1. repeated crystallization is easy to damage the product structure;
2. the double aqueous phase extraction has the advantages of less selectable extraction system, narrow separation range, difficult post-treatment, easy emulsification and difficult solvent recovery;
3. the macroporous adsorption resin has the problems of complex process, large solvent treatment capacity, low yield, poor process stability, incapability of ensuring product purity and the like, and restricts the large-scale production of high-quality products;
4. the supercritical carbon dioxide extraction method has the defects of higher extraction condition requirement, high cost and potential safety hazard at high temperature and high pressure;
in order to ensure the stable and controllable quality of the mono-ammonium glycyrrhizinate, the research on the glycyrrhizinate separation technology with application value is of great significance;
in recent years, the annual demand of liquorice in international markets is kept between 20 and 25 million tons (grade dry products), wherein high-specification liquorice is always in high-price. The annual total demand of China for liquorice is 6 to 7 million tons, wherein the liquorice products are consumed by the existing pharmaceutical industry about 1300 to 2000 tons each year.
The present invention is proposed in view of the above technical background and development prospects.
Disclosure of Invention
The principle of the method is that components in a mixture with similar structures are in a fixed phase by virtue of the adsorption effect of the surface of the fixed phase, the migration speeds of the components in the mixture along with the movement of a mobile phase are different, so that the components flow out of the fixed phase in a certain sequence, and a target fraction is picked up according to a chromatogram map to obtain a high-purity product.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
the invention relates to a method for separating and purifying glycyrrhizic acid extract by using preparative chromatography, which adopts a chromatographic separation mechanism, elutes the glycyrrhizic acid extract by using an organic solvent mixed solution, and collects glycyrrhizic acid target analysis solution.
The invention relates to a method for separating and purifying glycyrrhizic acid extract by using preparative chromatography, which is characterized by comprising the following steps: the special filler used in the chromatographic separation mode is silica gel modified by polar groups, a super-strong tail-sealing bonding mode is adopted, one or more of amino, diamino, amido, chlorine atom, bromine atom, cyano and phenyl are connected by C1-C30 n-alkyl groups, the number of the polar groups on each C1-C30 n-alkyl group connecting chain is more than 1, the molar ratio of the silicon hydroxyl on the surface of the activated silica gel to the aminosilane, diamino silane, amido silane, chloro silane, bromo silane, cyano silane and phenyl silane is 1: (0.6 to 1.0) and the bonding amount of the polar group is 0.6 to 6.0. mu. mol/m2The kind and bonding number of the bonding groups can be regulated and controlled, the particle diameter is 5-100 mu m, and the pore diameter is
The shape is irregular or spherical.
The invention relates to a method for separating and purifying glycyrrhizic acid extract by using preparative chromatography, which is characterized by comprising the following steps: the mobile phase is one or two (organic phase) and acid water (water phase) mixed, the organic phase is one or more of common solvents such as methanol, ethanol, isopropanol, acetonitrile, acetone and the like, and the water phase is one or two of formic acid, acetic acid and oxalic acid, and the proportion is 0.2-5.0% (v/v).
The invention relates to a method for separating and purifying glycyrrhizic acid extract by using preparative chromatography, which is characterized by comprising the following steps: the sample loading amount of the upper column adsorption is the ratio of the mass of the sample loading sample to the mass of the filler, and the sample loading amount is 1-12%.
The invention relates to a method for separating and purifying glycyrrhizic acid extract by using preparative chromatography, which is characterized by comprising the following steps: and collecting target analysis liquid according to the monitoring peak curve of the detector.
The invention relates to a method for separating and purifying glycyrrhizic acid extract by using preparative chromatography, which comprises the following steps:
1) weighing a glycyrrhizic acid extract sample, dissolving the glycyrrhizic acid extract sample by using a mixed solution of an organic solvent and pure water, wherein the organic solvent is one or more of common solvents such as acetone, methanol, ethanol, acetonitrile and the like, the proportion is 1-80% (v/v), and the concentration is 20-100 mg/mL to serve as a loading solution;
2) separating by adopting a column system, wherein the inner diameter of a chromatographic column is 10-1200 mm; a chromatographic separation and purification system is adopted, the filler is polar modified silica gel, the organic solvent is one or more of common solvents such as methanol, ethanol, isopropanol, acetonitrile and acetone, and the organic solvent accounts for 10-60% of the total volume of the mobile phase; the flow rate is 0.05 to 0.6 times the column volume/min. Collecting target analytical solution according to peak shape curve of detection spectrogram (collecting according to chromatographic peak), and obtaining qualified glycyrrhizic acid product (content is greater than 90%, related substance impurity A is less than 3%, isomer impurity 18 alpha glycyrrhizic acid is less than 5%).
The invention has the following advantages:
1. the glycyrrhizic acid extract is purified and prepared by using a preparative chromatographic separation and purification means and adopting the silica gel filler modified by polar groups, and compared with other separation processes such as traditional silica gel and resin, the glycyrrhizic acid extract has the advantages of rapidness and high efficiency;
2. the preparation process is adopted for separation and purification, the separation selectivity is good, the related impurity A and the isomer impurity 18 alpha-glycyrrhizic acid are effectively removed, and the product content is high;
3. the preparation process is adopted for separation and purification, the sample loading amount is large, the recovery rate is high, and the industrial production is easier to realize.
4. The preparation process is adopted for separation and purification, the solvent consumption is low, the flow is simple, the repeatability is good, the operation is simple and controllable, the automation is easy to realize, and the process is stable.
Drawings
FIG. 1 is a spectrum of glycyrrhizic acid extract preparation in example 1;
FIG. 2 is a liquid phase analysis spectrum of the purified glycyrrhizic acid product of example 1.
Detailed Description
The present invention will now be further described with reference to examples. It should be noted that, for those skilled in the art, without departing from the technical principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be construed as the protection scope of the present invention. The examples are given solely for the purpose of illustrating the invention and are not to be construed as limiting the invention thereto.
Example 1
Adding 15g of glycyrrhizic acid extract into 300mL of 50% acetone water for dissolving, and preparing sample loading liquid with the concentration of 50mg/mL, wherein the sample loading amount is 5%. The silica gel was modified with polar groups (C18 attached to diamino modified filler with a bond of 2.28. mu. mol/m2) Packed chromatographic column (column size 50X 250mm, particle size 30 μm, pore size
Filler mass 300 g). Using acetone: eluting with 1% acetic acid solution (v/v) at flow rate of 50mL/min 10:90, collecting the eluate according to peak profile curve for 30-98 min, concentrating and drying the target eluate by conventional method to obtain product glycyrrhizic acid with glycyrrhizic acid content of 91.76% (related substance impurity A2.51%, isomer impurity 18 α glycyrrhizic acid 3.11%), and yield of 93.8%.
Example 2
Dissolving glycyrrhizic acid extract 5g in 50mL 50% methanol water to obtain sample solution with concentration of 100mg/mL and sample loading amount of 10%. Polar group modified silica gel (C8 is connected with amido modified filler, bonding amount is 2.41 mu mol/m2) Packed chromatographic column (column size 20X 250mm, particle size 10 μm, pore size)
Filler mass 50 g). Using methanol: eluting with 0.5% formic acid solution (v/v) 20:80 at flow rate of 50mL/min, collecting the target peak according to the peak profile curve for 35-90 min, concentrating and drying the target analytic solution by the prior art to obtain the product with glycyrrhizic acid content of 93.25% (related substance impurity A2.08%, isomer impurity 18 alpha glycyrrhizic acid 2.86%) and yield of 92.5%.
Example 3
Dissolving 10kg glycyrrhizic acid extract in 100L 50% ethanol water to obtain sample loading solution with concentration of 100mg/mL, and loading the sample amount of 8%. The silica gel is modified by polar groups (C4 is connected with amino modified filler, and the bonding amount is 2.69 mu mol/m2) Packed chromatographic column (column size 1000X 250mm, particle size 30 μm, pore size)
Filler mass 120 kg). The reaction solution is prepared by using ethanol: eluting with oxalic acid water (2%, v/v) at a flow rate of 25:75 at 20L/min, collecting the eluate according to a peak profile curve for 32-98 min, concentrating and drying the target eluate by the prior art to obtain a product with glycyrrhizic acid content of 93.25% (related substance impurity A2.08%, isomer impurity 18 α glycyrrhizic acid 2.48%), and yield of 90.5%.
Comparative example 1
The difference from the example 1 is that bare silica gel (100-200 mesh) modified by non-bonding group is used as separation material, the other conditions are the same as the example 1, the glycyrrhizic acid content is 54.6% (related substance impurity A is 6.28%, isomer impurity 18 alpha glycyrrhizic acid is 9.17%), and the yield is 53.8%.
Comparative example 2
The difference from the example 1 is that macroporous resin is used as a separation material, the other conditions are the same as the example 1, the purity of the obtained glycyrrhizic acid is 63.3 percent (related substance impurity A is 8.21 percent, isomer impurity 18 alpha glycyrrhizic acid is 9.27 percent), and the yield is 62.2 percent.
Comparative example 3
The difference from the example 1 is that ordinary octadecylsilane chemically bonded silica is used as a separation material, the other conditions are the same as the example 1, and the glycyrrhizic acid content of the obtained product is 52.97% (related substance impurity A is 4.36%, isomer impurity 18 alpha glycyrrhizic acid is 10.59%) and the yield is 61.3%.
Claims (8)
1. A method for separating and purifying a liquorice extract by using a preparative chromatography is characterized by comprising the following steps: and (3) eluting by using an organic solvent as a mobile phase in a chromatographic separation mode, and collecting a target analytic solution to obtain a high-purity glycyrrhizic acid product (the content is more than 90 percent, and limited impurities are controlled in a qualified range).
2. The method of claim 1, wherein: dissolving the glycyrrhizic acid extract by using a mixed solution of an organic solvent and pure water, wherein the organic solvent is one or more of common solvents such as methanol, ethanol, isopropanol, acetonitrile, acetone and the like, the proportion is 1-90% (v/v), and the sample concentration is 10-200 mg/mL.
3. The method of claim 1, wherein: the filler used in the chromatographic separation mode is silica gel modified by polar groups, and is prepared by adopting a super-strong tail-sealing bonding mode, one or more of amino, diamino, amido, chlorine atom, bromine atom, cyano and phenyl polar groups are connected with the silica gel through C1-C30 n-alkane groups, the number of the polar groups on each C1-C30 n-alkane group connecting chain is more than 1, the molar ratio of the silicon hydroxyl on the surface of the activated silica gel to the aminosilane, diamino silane, amido silane, chlorine silane, bromine silane, cyano silane and phenyl silane is 1: (0.5 to 1.5) and the bonding amount of the polar group is 0.5 to 8.0. mu. mol/m2The types and bonding quantity of the bonding groups can be regulated and controlled.
4. The method of claim 1, wherein: the chromatographic packing has a particle size of 5-100 μm and a pore diameter of
The shape is irregular or spherical.
5. The method of claim 1, wherein: the mobile phase is one or two (organic phase) and acid water (water phase) mixed, the organic phase is one or more of common solvents such as methanol, ethanol, isopropanol, acetonitrile, acetone and the like, the organic phase accounts for 10-60% of the total volume of the mobile phase, the water phase is one or two of formic acid, acetic acid and oxalic acid, the proportion is 0.1-6.0% (v/v), and the type of the mobile phase is optimized and adjusted according to different bonding fillers.
6. The method of claim 1, wherein: the flow velocity of the mobile phase is 0.02-0.8 times of the column volume/min.
7. The method of claim 1, wherein: the sample loading amount of the upper column adsorption is the ratio of the sample loading mass to the filler mass, and the sample loading amount is 0.1-15%.
8. The method of claim 1, wherein: collecting target analysis solution according to chromatogram, concentrating and drying to obtain high purity glycyrrhizic acid product with content of more than 90% (related substance impurity A < 3%, isomer impurity 18 alpha glycyrrhizic acid < 5%).
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