CN117105999A - Method for separating and extracting arbutin by using simulated moving bed chromatography - Google Patents
Method for separating and extracting arbutin by using simulated moving bed chromatography Download PDFInfo
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- CN117105999A CN117105999A CN202311075138.3A CN202311075138A CN117105999A CN 117105999 A CN117105999 A CN 117105999A CN 202311075138 A CN202311075138 A CN 202311075138A CN 117105999 A CN117105999 A CN 117105999A
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- BJRNKVDFDLYUGJ-RMPHRYRLSA-N hydroquinone O-beta-D-glucopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-RMPHRYRLSA-N 0.000 title claims abstract description 180
- 229960000271 arbutin Drugs 0.000 title claims abstract description 89
- BJRNKVDFDLYUGJ-UHFFFAOYSA-N p-hydroxyphenyl beta-D-alloside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-UHFFFAOYSA-N 0.000 title claims abstract description 89
- 238000004587 chromatography analysis Methods 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 34
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 claims abstract description 88
- PVXPPJIGRGXGCY-TZLCEDOOSA-N 6-O-alpha-D-glucopyranosyl-D-fructofuranose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)C(O)(CO)O1 PVXPPJIGRGXGCY-TZLCEDOOSA-N 0.000 claims abstract description 49
- 238000006243 chemical reaction Methods 0.000 claims abstract description 33
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 33
- 238000000926 separation method Methods 0.000 claims abstract description 33
- 238000000605 extraction Methods 0.000 claims abstract description 24
- 239000007788 liquid Substances 0.000 claims abstract description 21
- 239000003480 eluent Substances 0.000 claims description 78
- 239000002994 raw material Substances 0.000 claims description 60
- 230000014759 maintenance of location Effects 0.000 claims description 24
- 230000005526 G1 to G0 transition Effects 0.000 claims description 21
- 239000012528 membrane Substances 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 238000013375 chromatographic separation Methods 0.000 claims description 14
- 239000003729 cation exchange resin Substances 0.000 claims description 12
- 239000008367 deionised water Substances 0.000 claims description 12
- 229910021641 deionized water Inorganic materials 0.000 claims description 12
- 238000001914 filtration Methods 0.000 claims description 12
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 11
- 239000011591 potassium Substances 0.000 claims description 11
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 10
- 229910052700 potassium Inorganic materials 0.000 claims description 10
- 238000007599 discharging Methods 0.000 claims description 8
- 238000001728 nano-filtration Methods 0.000 claims description 6
- 238000005192 partition Methods 0.000 claims description 5
- 239000011734 sodium Substances 0.000 claims description 5
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 4
- 229910052708 sodium Inorganic materials 0.000 claims description 4
- 238000005374 membrane filtration Methods 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 2
- 238000011084 recovery Methods 0.000 abstract description 7
- 239000000243 solution Substances 0.000 description 67
- 239000011347 resin Substances 0.000 description 15
- 229920005989 resin Polymers 0.000 description 15
- BJRNKVDFDLYUGJ-ZIQFBCGOSA-N alpha-Arbutin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-ZIQFBCGOSA-N 0.000 description 13
- 229940033280 alpha-arbutin Drugs 0.000 description 13
- 230000000694 effects Effects 0.000 description 8
- 239000002245 particle Substances 0.000 description 7
- 241001052560 Thallis Species 0.000 description 6
- 239000012535 impurity Substances 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 239000003463 adsorbent Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 230000002087 whitening effect Effects 0.000 description 4
- 102000003425 Tyrosinase Human genes 0.000 description 3
- 108060008724 Tyrosinase Proteins 0.000 description 3
- 238000002425 crystallisation Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 239000000945 filler Substances 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- DLRVVLDZNNYCBX-RTPHMHGBSA-N isomaltose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 DLRVVLDZNNYCBX-RTPHMHGBSA-N 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical group [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
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- 150000007949 saponins Chemical class 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 1
- 240000004972 Bergenia crassifolia Species 0.000 description 1
- 235000014785 Bergenia crassifolia Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 240000003377 Shepherdia canadensis Species 0.000 description 1
- 235000018324 Shepherdia canadensis Nutrition 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 240000000851 Vaccinium corymbosum Species 0.000 description 1
- 235000003095 Vaccinium corymbosum Nutrition 0.000 description 1
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- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000021014 blueberries Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
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- 230000007547 defect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
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- 239000012895 dilution Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- GCFHZZWXZLABBL-UHFFFAOYSA-N ethanol;hexane Chemical compound CCO.CCCCCC GCFHZZWXZLABBL-UHFFFAOYSA-N 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 238000013486 operation strategy Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000002952 polymeric resin Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 229920003002 synthetic resin Polymers 0.000 description 1
- GYDJEQRTZSCIOI-LJGSYFOKSA-N tranexamic acid Chemical class NC[C@H]1CC[C@H](C(O)=O)CC1 GYDJEQRTZSCIOI-LJGSYFOKSA-N 0.000 description 1
- 238000006276 transfer reaction Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
- C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
- C07C37/685—Processes comprising at least two steps in series
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
- C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
- C07C37/70—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment
- C07C37/82—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment by solid-liquid treatment; by chemisorption
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/04—Disaccharides
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a method for separating and extracting arbutin by utilizing simulated moving bed chromatography. According to the method, the arbutin is efficiently separated from the enzymatic conversion liquid through the simulated moving bed, so that the one-time separation and extraction of the arbutin can be realized, the yield and purity of the arbutin are improved, isomaltulose and hydroquinone in the enzymatic conversion liquid can be simultaneously recovered, and the value of each component in the enzymatic conversion liquid is fully excavated. The purity and recovery rate of isomaltulose in the product can reach more than 98% and more than 99%, the purity and recovery rate of arbutin can reach more than 99% and more than 93.9%, the purity and recovery rate of hydroquinone can reach more than 94% and more than 95%, respectively, and the product has higher purity and yield.
Description
Technical Field
The invention relates to the technical field of biochemical separation, in particular to a method for separating and extracting arbutin by utilizing simulated moving bed chromatography.
Background
Arbutin (Arbutin) is also called Arbutin and Arbutin, belongs to glucoside of hydroquinone (hydroquinone), and is an emerging natural whitening active substance with strong compatibility, no allergy and no irritation. The arbutin has the effect of inhibiting the activity of tyrosinase, can reduce the deposition of the tyrosinase in skin, can effectively whiten the skin, and has good market prospect. The arbutin can be matched with various products for use, for example, the arbutin, derivatives of tranexamic acid, esculentol and the like have excellent whitening effect after being matched, and the ascorbic acid, the arbutin phosphate and the like have ideal whitening effect. The arbutin has two isomers, namely alpha-arbutin and beta-arbutin. From the perspective of whitening mechanism, alpha-arbutin only inhibits tyrosinase activity, but does not inhibit human cells; beta-arbutin can inhibit human cells and has certain side effects on users.
The synthesis of arbutin is mainly divided into three categories, namely plant extraction, organic synthesis and bioconversion. Currently, α -arbutin is mainly prepared by performing a sugar transfer reaction or a reverse hydrolysis reaction by a biological method. Zhou Qi (food and fermentation industries, 2021,47 (22)) 1-7, wherein hydroquinone and sucrose are used as substrates by bioconversion, sucrose phosphorylase is used as biocatalyst and is optimized by bacillus subtilis to obtain whole cell catalysis, the yield of the optimized alpha-arbutin reaches 119.44g/L, and the molar conversion rate of the substrates is 96.56%.
The method for separating alpha-arbutin mainly comprises membrane separation method, solvent extraction method, crystallization method, chromatography, etc. The membrane separation method is to remove thalli, larger proteins and impurity particles from the alpha-arbutin fermentation liquor through membrane filtration, and then to prepare the alpha-arbutin by combining other methods. Zheng Xiongmin (food and fermentation industry, 2015,41 (06): 104-107) passing the fermentation broth of alpha-arbutin through ceramic filter membrane, adsorbing and desorbing with DA201 macroporous adsorbent resin and D315 anion exchange resin, and finally obtaining crystalline alpha-arbutin product. The solvent extraction method is a method for separating by using a similar compatible principle and selecting a solution with polarity similar to that of the alpha-arbutin as a solvent. Zheng Linlu (Probex of the chemical society, 2021,41 (02): 145-147) is used for extracting soapberry saponin with ethanol and surfactant, and the extraction rate of total saponin reaches 69.9%. Crystallization methods make use of the separation of components of a mixture that differ in solubility in the same solvent or in solubility at different temperatures. The crystallization method has large solvent consumption in purifying the glycoside substances, which affects the application of the method to a certain extent.
The chromatographic separation method is an efficient separation and purification method, and the polymer resin used as the chromatographic filler has adjustable particle size, porosity and functional groups, and can achieve good analysis effect by selecting proper filler. Liu Min et al (Chinese patent medicine, 2013,35 (4): 800-803) analyzed and determined arbutin in bergenia crassifolia tissue culture regeneration seedlings by using a C18 chromatographic column. Zhang Le et al (daily chemical industry, 2015,45 (1): 55-57+59) used S-Chiral A chromatographic column, and n-hexane-ethanol mixed solution as eluent to perform binary gradient elution, and perform qualitative analysis and accurate quantitative detection on two kinds of arbutin in cosmetics. In addition, chromatography has been widely used for separation, analysis and purification of saccharides and glycosides. The separation and purification of alpha-arbutin mainly uses macroporous adsorption resin as filler. Liu Chunqiao and 2005 (06) 364-367+383) are prepared by fermenting with para-diphenol and sucrose as reactants, and S-8 polar resin is selected to adsorb alpha-arbutin, and after crystallization and purification, the purity of the obtained alpha-arbutin is up to 99%, and the yield is above 85%. Zhang Wenlei et al (food and fermentation industries, 2017,43 (6): 49-53) used AB-8 macroporous adsorbent resin to separate and purify the enzymatically synthesized alpha-arbutin, eluted with 30% ethanol solution with a recovery of 88%.
With the deep research of chromatography, the preparative chromatographic separation and purification technology is found to be an effective means for obtaining a large amount of high-value or high-activity components. Simulated moving bed chromatography is a continuous preparative chromatographic separation technique that combines the advantages of a fixed bed and a true moving bed, such as fractionationThe simulated moving bed chromatography has strong separation capability, less eluent consumption, no abrasion of a bed layer, continuous production and the like, is widely applied to a plurality of industries and fields in recent years, and achieves good results. Chinese patent CN2484564 provides a simulated moving bed apparatus of open loop structure, which is a loop structure with head and tail ends and moving periodically, formed by serially connecting a plurality of chromatographic columns filled with adsorbent, and is suitable for separating enantiomers selectively under higher working pressure, wherein the chromatographic packing is a fine-particle chiral stationary phase. Chinese patent CN106267894a provides a series simulated moving bed system suitable for separating components with intermediate retention capacity from a mixture of three components; the series simulated moving bed system comprises a first stage simulated moving bed and a second stage simulated moving bed, can provide a side flow with stable concentration, continuously and stably separate out intermediate retention components, and can timely discharge components with strong retention property out of the system. In addition, chinese patent CN104672286a reports a method for enriching and purifying α -arbutin from blueberry leaves by four-zone simulated moving bed chromatography, and D101 macroporous adsorption resin is used as an adsorbent to separate and purify α -arbutin; each zone consists of 1-2C 18 chromatographic columns connected in series, the obtained solution is concentrated and crystallized, and the purity of the alpha-arbutin reaches more than 99 percent. Chinese patent CN103555794a discloses a clean production method of isomaltooligosaccharide, in which a sequential simulated moving bed is used to separate the concentrated solution, a strong acid resin is used as a stationary phase, effluent of isomaltooligosaccharide, glucose and polysaccharide are collected respectively, and the isomaltooligosaccharide effluent is concentrated and dried to obtain the product. Chinese patent CN110746469a discloses a method for separating isomaltulose mother liquor by a simulated moving bed; adding water into isomaltulose mother liquor for dilution, and obtaining supernatant after decoloring, centrifuging and membrane filtration concentration in sequence; separating the supernatant with simulated moving bed, and selecting K as adsorbent + 、Ca 2+ Or Na (or) + The strong acid cation exchange resin has the operation temperature of 50-60 ℃, the flow rate of feed liquid of 60-80 mL/min, the flow rate of eluent of 100-150 mL/min, the valve switching time of 300-480 s, the operation for 4-7 cycles reaches the equilibrium state, the extraction port and the raffinate port solution are collected, and the purity of the obtained isomaltulose can reach 98.7 percent.
In summary, the three-zone simulated moving bed is an improved chromatographic separation system, and has three chromatographic functional partitions, namely an extraction zone, an enrichment zone and a separation zone, and can effectively avoid pollution of liquid reflux to the extraction zone in the operation process because of no circulation zone. In addition, the three-zone simulated moving bed system can only realize separation of two components, the scheme improves the three-zone simulated moving bed device system, the proposed system architecture and operation strategy can separate and extract intermediate components in mixed liquid, and simultaneously, the steps of chromatographic separation operation are reduced, and the chromatographic separation efficiency of the simulated moving bed is greatly improved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for separating and extracting arbutin by utilizing simulated moving bed chromatography, and the arbutin can be efficiently separated from enzymatic conversion liquid by the simulated moving bed.
The technical scheme of the invention is as follows:
a method for separating and extracting arbutin by using simulated moving bed chromatography, comprising the following steps:
(1) Pretreatment of enzymatic conversion solution to obtain a raw material;
(2) Constructing a simulated moving bed chromatographic system;
(3) Simulated moving bed chromatographic separation: introducing the raw material obtained in the step (1) into a simulated moving bed chromatographic system, and separating to prepare arbutin;
in the step (2), the simulated moving bed chromatography comprises a zone I, a zone II, a zone III, a zone IV, a zone V and a zone VI, wherein the zone I, the zone II and the zone III are connected in sequence, the zone IV, the zone V and the zone VI are connected in sequence, and the zone III is disconnected from the zone IV in the initial stage; each zone of the simulated moving bed at least comprises 1 chromatographic column; the chromatograph of the simulated moving bed is divided into a separation area, an enrichment area and an extraction area according to the functional partition; the separation zone is located between the feed inlet and the raffinate outlet; the enrichment area is positioned between the extracting solution outlet and the raw material inlet; the extraction zone is located between the eluent inlet and the extract outlet.
Further, in the step (1), the specific method for pretreatment of the enzymatic conversion solution is as follows: filtering the crude enzymatic conversion solution by a membrane, and concentrating to obtain a raw material; the membrane filtration means filtration by using a nanofiltration membrane, wherein the aperture of the nanofiltration membrane is 50-1000 nm, the filtration pressure is 0.2-0.4 MPa, and the temperature is 30-50 ℃; the concentration refers to concentrating the enzymatic conversion solution filtered by the nanofiltration membrane until the mass concentration is 40% -60%; the raw materials comprise the following components in percentage by mass: the content of arbutin is 40-70%, the content of hydroquinone is 20-40%, and the content of isomaltulose is 10-20%.
Further, in the step (2), the simulated moving bed chromatography system uses potassium type or sodium type cation exchange resin as a stationary phase and deionized water as an eluent.
Further, the flow rate of the eluent is: before feeding, flowing an eluent into the system, keeping the flow at 4-5 mL/min, stopping the operation of other pumps, and removing the gas remained in the column; the eluent flow rate is increased to a target value of 6-7 mL/min in the separation zone.
Further, in the step (2), the simulated moving bed chromatography system needs to keep the temperature of the chromatographic column at 60-80 ℃ in operation.
Further, in the step (2), the simulated moving bed chromatography system further comprises a first eluent inlet, a first extract outlet, a raw material inlet, a first raffinate outlet, a second eluent inlet, a second extract outlet, a first raffinate inlet, and a second raffinate outlet;
the area I is positioned between the first eluent inlet and the first extracting solution outlet; the zone II is positioned between the first extracting solution outlet and the raw material inlet; the zone III is located between the feed inlet and the first raffinate outlet; the IV zone is positioned between the second eluent inlet and the second extracting solution outlet; the V zone is positioned between the second extract outlet and the first raffinate inlet; the VI zone is located between the first raffinate inlet and the second raffinate outlet;
the separation zone is located between the feed inlet and the raffinate outlet; the enrichment zone is positioned between the extracting solution outlet and the raw material inlet; the extraction zone is located between the eluent inlet and the extract outlet.
Further, the first extract contains hydroquinone; the second extracting solution contains arbutin; the first raffinate contains arbutin and isomaltulose; the second raffinate contains isomaltulose.
Further, in step (2), the simulated moving bed chromatography system further comprises an eluent inlet valve, an extracting solution valve, a raffinate valve and a feed valve.
Further, in the step (3), the specific steps of the simulated moving bed chromatographic separation are as follows:
firstly, opening an eluent inlet valve in front of a 1 st chromatographic column in an area I and an eluent inlet valve in front of a 1 st chromatographic column in an area IV and an extraction valve in front of the 1 st chromatographic column in an area II, and then opening a feed valve in front of the 1 st chromatographic column in an area III to input raw material liquid into a system, wherein the feed flow rate of the raw material liquid is 3-5 mL/min, and performing chromatographic separation;
eluent is respectively input into the I zone and the IV zone, and the first raffinate flowing out of the III zone flows to a raffinate inlet between the V zone and the VI zone through a pipeline; the hydroquinone of the first extracting solution flows out between the last 1 chromatographic column of the zone I and the 1 st chromatographic column of the zone II; the tail of the last 1 chromatographic column in the III zone flows out of the first raffinate arbutin and isomaltulose; the tail of the last 1 chromatographic column in the IV zone flows out of the arbutin of the second extracting solution; the tail of the last 1 chromatographic column in the VI zone flows out of the isomaltulose of the second raffinate;
after the eluent inlet, the raw material inlet, the extracting solution outlet and the raffinate outlet run for 1 switching period, the eluent inlet, the raw material inlet, the extracting solution outlet and the raffinate outlet are synchronously switched to the next sequential position along the flowing direction of the eluent, the disconnected position of the simulated moving bed partition is synchronously switched to the next sequential position along the flowing direction of the eluent, the initial position of the inlet and outlet valves is recovered after the running circulation of all the inlet and outlet valves is completed, the running steps are repeated, and medium retention component arbutin with higher purity can be continuously obtained.
Further, in the step (3), there are two sub-periods within the 1 switching period, and the two sub-periods are specifically as follows:
(a) Sub-period one: firstly, separating hydroquinone components from raw materials for 10-16 min;
(b) Sub-period two: and separating the arbutin component from the first raffinate for 8-12 min.
The working mechanism of the invention is as follows: the hydroxyl groups on the hydroquinone molecule carry a very weak negative charge; the hydroxyl groups on the sugar molecules or arbutin molecules are also very weakly negatively charged, and the hydroxyl groups on their anomeric carbons can be deprotonated to have a stronger negative charge. Therefore, the negative charge of hydroquinone molecules or sugar molecules or arbutin molecules and the positive charge of potassium or sodium ions on the surface of the resin generate an affinity force due to the electrostatic neutralization; the stronger the negative charge of the molecule, the stronger its affinity, and the longer the retention time. However, arbutin molecules and isomaltulose molecules have a relatively large molecular weight, and when larger than the size of the resin pore canal, produce a size exclusion effect, and the corresponding retention time on the chromatographic column is reduced. The retention time of isomaltulose molecules in single column chromatography is short, the retention time of arbutin is inferior, and the retention time of hydroquinone is longest. Inputting refined enzymatic conversion solution into a simulated moving bed chromatographic system, collecting hydroquinone with strong retention component, inputting the separated mixed solution of arbutin and isomaltulose into the simulated moving bed chromatographic system again, and collecting arbutin with medium retention component and isomaltulose with weak retention component. Ports are switched along the flowing direction of the eluent to simulate the movement of a stationary phase, and the arbutin can be continuously and efficiently separated and extracted.
The beneficial technical effects of the invention are as follows:
(1) The simulated moving bed can realize one-time separation and extraction of arbutin, improves the yield and purity of the arbutin, can simultaneously recover isomaltulose and hydroquinone in enzymatic conversion liquid, and fully excavates the value of each component in the enzymatic conversion liquid. The purity and recovery rate of isomaltulose in the product can reach more than 98% and more than 99%, the purity and recovery rate of arbutin can reach more than 99% and more than 93.9%, the purity and recovery rate of hydroquinone can reach more than 94% and more than 95%, respectively, and the product has higher purity and yield.
(2) The invention adopts simulated moving bed chromatography to separate arbutin, reduces the resin consumption of stationary phase and the number of chromatographic columns, reduces investment cost and material consumption, simplifies operation steps and control systems, and solves the pollution problem of separation functional areas due to no circulating area.
Drawings
FIG. 1 is a schematic diagram of the invention for separating and extracting arbutin by simulated moving bed chromatography.
FIG. 2 is a schematic diagram of five-zone conventional simulated moving bed separation and extraction of arbutin.
FIG. 3 is a schematic diagram of the separation and extraction of arbutin by a multi-column batch simulated moving bed.
Detailed Description
The present invention will be described in detail below with reference to the drawings and examples.
As shown in FIG. 1, the simulated moving bed in the following embodiment is formed by sequentially connecting a zone I, a zone II, a zone III, a zone IV, a zone V and a zone VI in series, wherein the zone I, the zone II and the zone III are sequentially connected, the zone IV, the zone V and the zone VI are sequentially connected, the zone III is disconnected from the zone IV, each zone at least comprises 1 chromatographic column and is divided into a separation zone, an enrichment zone and an extraction zone; the adjacent chromatographic columns are connected through a pipeline, and a one-way valve is arranged on the pipeline; the chromatographic column needs to be insulated at the temperature of 60-80 ℃. The simulated moving bed chromatography uses potassium type or sodium type cation exchange resin as a stationary phase, uses water as an eluent, and the eluent enters a chromatography system through a water inlet pipeline;
the simulated moving bed chromatography consists of a zone I, a zone II, a zone III, a zone IV, a zone V and a zone VI which are sequentially connected in series; each zone of the simulated moving bed at least comprises 1 chromatographic column, and is divided into a separation zone, an enrichment zone and an extraction zone; when the simulated moving bed chromatographic system is in operation, the zone I is positioned between the first eluent inlet and the first extracting solution outlet; the zone II is positioned between the first extracting solution outlet and the raw material inlet; zone III is located between the feed inlet and the first raffinate outlet; zone IV is located between the second eluent inlet and the second extract outlet; the V zone is positioned between the second extract outlet and the first raffinate inlet; zone VI is located between the first raffinate inlet and the second raffinate outlet; the first extracting solution contains hydroquinone with strong retention component; the second extract contains medium retention component arbutin; the first raffinate contains medium retention component arbutin and weak retention component isomaltulose; the second raffinate contains a weak retention component isomaltulose.
Taking 1 chromatographic column as an example in each zone, opening the eluent inlet valves in front of the No. 1 chromatographic column and in front of the No. 4 chromatographic column and the extraction valve of the No. 2 chromatographic column, respectively inputting the eluent into the zone I and the zone IV, and enabling the first raffinate to flow to a raffinate inlet between the zone V and the zone VI through a pipeline; starting a feed valve in front of a No. 3 chromatographic column to input raw material liquid into the system, and running chromatographic separation; a first extracting solution, namely the hydroquinone which is a strong retention component, flows out between the chromatographic column 1 and the chromatographic column 2; the tail of the No. 3 chromatographic column flows out of the first raffinate, namely medium retention component arbutin and weak retention component isomaltulose; the tail of the No. 4 chromatographic column flows out medium retention component arbutin; the fraction isomaltulose with weak retention was eluted from the tail of column 6. After the eluent inlet, the raw material inlet, the extracting solution outlet and the raffinate outlet run out in 1 switching period, the eluent inlet, the raw material inlet, the extracting solution outlet and the raffinate outlet are synchronously switched to the next sequential position along the liquid flowing direction, and medium retention component arbutin with higher purity can be continuously obtained.
The specific switching is performed in comparison with that shown in fig. 1, in the nth step, i.e. at the beginning of a cycle, the zone I, the zone II and the zone III are connected in sequence, the zone IV, the zone V and the zone VI are connected in sequence, and the zone III is disconnected from the zone IV, wherein the zone I is located between the first eluent inlet and the first raffinate outlet, the zone II is located between the first eluent outlet and the raw material inlet, the zone III is located between the raw material inlet and the first raffinate outlet, the zone IV is located between the second eluent inlet and the second extract outlet, the zone V is located between the second extract outlet and the first raffinate inlet, and the zone VI is located between the first raffinate inlet and the second raffinate outlet; in the n+1th step, the zone II, the zone III and the zone IV are sequentially connected, the zone V, the zone VI and the zone I are sequentially connected, and the zone IV is disconnected from the zone V, wherein the zone II is positioned between the first eluent inlet and the first extracting solution outlet, the zone III is positioned between the first extracting solution outlet and the raw material inlet, the zone IV is positioned between the raw material inlet and the first raffinate outlet, the zone V is positioned between the second eluent inlet and the second extracting solution outlet, the zone VI is positioned between the second extracting solution outlet and the first raffinate inlet, and the zone I is positioned between the first raffinate inlet and the second raffinate outlet; in the n+2 step, the III region, the IV region and the V region are sequentially connected, the VI region, the I region and the II region are sequentially connected, and the V region is disconnected from the VI region, wherein the III region is positioned between the first eluent inlet and the first extracting solution outlet, the IV region is positioned between the first extracting solution outlet and the raw material inlet, the V region is positioned between the raw material inlet and the first raffinate outlet, the VI region is positioned between the second eluent inlet and the second extracting solution outlet, the I region is positioned between the second extracting solution outlet and the first raffinate inlet, and the II region is positioned between the first raffinate inlet and the second raffinate outlet; in the n+3 step, the IV area, the V area and the VI area are sequentially connected, the I area, the II area and the III area are sequentially connected, and the VI area is disconnected from the I area, wherein the IV area is positioned between the first eluent inlet and the first extracting solution outlet, the V area is positioned between the first extracting solution outlet and the raw material inlet, the VI area is positioned between the raw material inlet and the first raffinate outlet, the I area is positioned between the second eluent inlet and the second extracting solution outlet, the II area is positioned between the second extracting solution outlet and the first raffinate inlet, and the III area is positioned between the first raffinate inlet and the second raffinate outlet; in the step N+4, the V area is sequentially connected with the VI area and the I area, the II area, the III area and the IV area are sequentially connected, and the I area is disconnected with the II area, wherein the V area is positioned between the first eluent inlet and the first extracting solution outlet, the VI area is positioned between the first extracting solution outlet and the raw material inlet, the I area is positioned between the raw material inlet and the first raffinate outlet, the II area is positioned between the second eluent inlet and the second extracting solution outlet, the III area is positioned between the second extracting solution outlet and the first raffinate inlet, and the IV area is positioned between the first raffinate inlet and the second raffinate outlet; in the n+5 step, the VI zone, the I zone, and the II zone are connected in sequence, the III zone, the IV zone, and the V zone are connected in sequence, and the II zone is disconnected from the III zone, wherein the VI zone is located between the first eluent inlet and the first extract outlet, the I zone is located between the first extract outlet and the feedstock inlet, the II zone is located between the feedstock inlet and the first raffinate outlet, the III zone is located between the second eluent inlet and the second extract outlet, the IV zone is located between the second extract outlet and the first raffinate inlet, and the V zone is located between the first raffinate inlet and the second raffinate outlet; after all the steps are completed, the valve is restored to the initial position of the inlet valve and the outlet valve, and the operation steps are repeated.
The "about" mentioned in the examples below is in the range of the numerical percentages given + -2%.
Example 1
A method for separating and extracting arbutin by using a simulated moving bed, the extraction flow of which is shown in figure 1, specifically comprises the following steps:
(1) Pretreatment of enzymatic conversion solution: filtering the crude enzymatic conversion solution by a filter membrane with the membrane aperture of 500nm, controlling the temperature at 40 ℃ and the pressure at 0.2MPa, removing thalli, larger proteins and impurity particles, concentrating the solution to the mass concentration of about 55%, and obtaining refined enzymatic conversion solution, namely the raw material; the raw materials comprise about 60% of arbutin, about 30% of hydroquinone and about 10% of isomaltulose by mass percent.
(2) Constructing simulated moving bed chromatography: keeping the temperature of the chromatographic column to 60 ℃, and taking potassium-type or sodium-type cation exchange resin as a stationary phase; deionized water is used as an eluent, and the running temperature is 60 ℃; before feeding, deionized water at 60 ℃ is flowed into the system as an eluent, the flow is kept at 4mL/min, and the operation of other pumps is stopped at the same time, so that the gas remained in the column is discharged; the separation zone increased the eluent flow to a target value of 6mL/min.
(3) Simulated moving bed chromatographic separation: according to the difference of affinity of the potassium stationary phase resin to different components, a feed port and a discharge port are configured, three components of hydroquinone, arbutin and isomaltulose in raw materials are separated and extracted according to the steps, the feed flow rate of the raw materials is 3mL/min, after all the steps are operated, the positions of all the feed and discharge valves are moved forward by one chromatographic column along the liquid flow direction, the initial positions of the feed and discharge valves are recovered after all the feed and discharge valves are operated and circulated, the steps are repeatedly operated, and the isomaltulose component, the arbutin component and the hydroquinone component are respectively collected.
Repeating the steps, and after 15 times of circulation, the yield of isomaltulose is about 98.8%, the purity is about 98.2%, the yield of arbutin is about 93.9%, and the purity is about 99.6%; the yield of hydroquinone is about 94.8% and the purity is about 95.7%.
Example 2
A method for separating and extracting arbutin by using a simulated moving bed, the extraction flow of which is shown in figure 1, specifically comprises the following steps:
(1) Pretreatment of enzymatic conversion solution: filtering the crude enzymatic conversion solution by a filter membrane with the membrane aperture of 500nm, controlling the temperature at 45 ℃ and the pressure at 0.3MPa, removing thalli, larger proteins and impurity particles, concentrating the solution to the mass concentration of about 50%, and obtaining refined enzymatic conversion solution, namely the raw material; the raw materials comprise about 50% of arbutin, about 40% of hydroquinone and about 10% of isomaltulose by mass percent.
(2) Constructing simulated moving bed chromatography: keeping the temperature of the chromatographic column to 65 ℃, and taking potassium-type or sodium-type cation exchange resin as a stationary phase; deionized water is used as an eluent, and the running temperature is 65 ℃; before feeding, deionized water at 65 ℃ is flowed into the system as an eluent, the flow is kept at 5mL/min, and the operation of other pumps is stopped at the same time, so that the gas remained in the column is discharged; the separation zone increased the eluent flow to a target value of 7mL/min.
(3) Simulated moving bed separation: according to the difference of affinity of the potassium stationary phase resin to different components, a feed port and a discharge port are configured, three components of hydroquinone, arbutin and isomaltulose in raw materials are separated and extracted according to steps, the feed flow rate of the raw materials is 4mL/min, after all the steps are operated, the positions of all the feed and discharge valves are moved forward by one chromatographic column along the liquid flow direction, the initial positions of the feed and discharge valves are recovered after all the operation cycles of the feed and discharge valves are completed, the operation steps are repeated, and the isomaltulose component, the arbutin component and the hydroquinone component are respectively collected.
Repeating the steps, and after 20 times of circulation, the yield of isomaltulose is about 99%, the purity is about 98%, the yield of arbutin is about 94%, and the purity is about 98%; the yield of hydroquinone is about 94% and the purity is about 95%.
Example 3
A method for separating and extracting arbutin by using a simulated moving bed, the extraction flow of which is shown in figure 1, specifically comprises the following steps:
(1) Pretreatment of enzymatic conversion solution: filtering the crude enzymatic conversion solution by a filter membrane with the membrane aperture of 50nm, controlling the temperature to be 50 ℃ and the pressure to be 0.4MPa, removing thalli, larger proteins and impurity particles, concentrating the solution to the mass concentration of about 60%, and obtaining refined enzymatic conversion solution, namely the raw material; the raw materials comprise about 50% of arbutin, about 35% of hydroquinone and about 15% of isomaltulose by mass percent.
(2) Constructing simulated moving bed chromatography: keeping the temperature of the chromatographic column to 75 ℃, and taking potassium-type or sodium-type cation exchange resin as a stationary phase; deionized water is used as an eluent, and the running temperature is 75 ℃; before feeding, deionized water at 75 ℃ is flowed into the system as an eluent, the flow is kept at 4mL/min, and the operation of other pumps is stopped at the same time, so that the gas remained in the column is discharged; the separation zone increased the eluent flow to a target value of 7mL/min.
(3) Simulated moving bed separation: according to the difference of affinity of sodium stationary phase resin to different components, a feed port and a discharge port are configured, three components of hydroquinone, arbutin and isomaltulose in raw materials are separated and extracted according to steps, the feed flow rate of the raw materials is 5mL/min, after all the steps are operated, the positions of all the feed and discharge valves are moved forward by one chromatographic column along the liquid flow direction, the initial positions of the feed and discharge valves are recovered after all the operation cycles of the feed and discharge valves are completed, the operation steps are repeated, and the isomaltulose component, the arbutin component and the hydroquinone component are respectively collected.
Repeating the steps, and after 25 times of circulation, the yield of isomaltulose is about 98%, the purity is about 97%, the yield of arbutin is about 93%, and the purity is about 97%; the yield of hydroquinone is about 93% and the purity is about 94%.
Example 4
A method for separating and extracting arbutin by using a simulated moving bed, the extraction flow of which is shown in figure 1, specifically comprises the following steps:
(1) Pretreatment of enzymatic conversion solution: filtering the crude enzymatic conversion solution by a filter membrane with the membrane aperture of 1000nm, controlling the temperature to be 30 ℃ and the pressure to be 0.1MPa, removing thalli, larger proteins and impurity particles, concentrating the solution to the mass concentration of about 40%, and obtaining refined enzymatic conversion solution, namely the raw material; the raw materials comprise about 60% of arbutin, about 25% of hydroquinone and about 15% of isomaltulose by mass percent.
(2) Constructing simulated moving bed chromatography: keeping the temperature of the chromatographic column to 80 ℃, and taking potassium-type or sodium-type cation exchange resin as a stationary phase; deionized water is used as an eluent, and the running temperature is 80 ℃; before feeding, deionized water at 80 ℃ is flowed into the system as an eluent, the flow is kept at 4mL/min, and the operation of other pumps is stopped at the same time, so that the gas remained in the column is discharged; the separation zone increased the eluent flow to a target value of 6mL/min.
(2) Simulated moving bed separation: according to the difference of affinity of the potassium stationary phase resin to different components, a feed port and a discharge port are configured, three components of hydroquinone, arbutin and isomaltulose in raw materials are separated and extracted according to the steps, the feed flow rate of the raw materials is 3mL/min, after all the steps are operated, the positions of all the feed and discharge valves are moved forward by one chromatographic column along the liquid flow direction, the initial positions of the feed and discharge valves are recovered after all the feed and discharge valves are operated and circulated, the steps are repeatedly operated, and the isomaltulose component, the arbutin component and the hydroquinone component are respectively collected.
Repeating the steps, and after 20 times of circulation, the yield of isomaltulose is about 98%, the purity is about 98%, the yield of arbutin is about 93%, and the purity is about 97%; the yield of hydroquinone is about 94% and the purity is about 94%.
Comparative example 1
A method for separating and extracting arbutin by a five-zone conventional simulated moving bed is shown in figure 2, and specifically comprises the following steps:
(1) Pretreatment of enzymatic conversion solution: filtering the crude enzymatic conversion solution by a filter membrane with the membrane aperture of 500nm, controlling the temperature at 40 ℃ and the pressure at 0.2MPa, removing thalli, larger proteins and impurity particles, concentrating the solution to the mass concentration of about 55%, and obtaining refined enzymatic conversion solution, namely the raw material; the raw materials comprise about 60% of arbutin, about 30% of hydroquinone and about 10% of isomaltulose by mass percent.
(2) Five-zone conventional simulated moving bed separation: according to the difference of affinities of the potassium stationary phase resin to different components, a feed port and a discharge port are configured, and three components of hydroquinone, arbutin and isomaltulose in the raw materials are separated and extracted according to the steps.
The chromatographic column needs to be insulated at 60 ℃; the five-zone conventional simulated moving bed chromatography uses potassium cation exchange resin as a stationary phase, water as an eluent, and the running temperature is 60 ℃; the stationary phase is potassium type cation exchange resin.
Before feeding, deionized water at 60 ℃ is fed into the five-zone conventional simulated moving bed chromatographic system as an eluent, the flow rate is kept at 4mL/min, the operation of other pumps is stopped, and the gas remained in the column is discharged. The separation zone increased the eluent flow to a target value of 6mL/min and the feed flow was 3mL/min.
After all the steps are finished, the position of each feeding and discharging valve is moved forward by one chromatographic column along the liquid flow direction, the initial position of the feeding and discharging valves is recovered after the operation cycle of all the feeding and discharging valves is finished, the steps are repeatedly operated, and isomaltulose components, arbutin components and hydroquinone components are respectively collected.
Repeating the steps, and after 15 times of circulation, the yield of isomaltulose is about 95%, the purity is about 95%, the yield of arbutin is about 91%, and the purity is about 94%; the yield of hydroquinone is about 92% and the purity is about 93%.
Comparative example 2
A method for separating and extracting arbutin by a multi-column intermittent simulated moving bed, the extraction flow is shown in figure 3, and specifically comprises the following steps:
(1) Pretreatment of enzymatic conversion solution: filtering the crude enzymatic conversion solution by a filter membrane with the membrane aperture of 500nm, controlling the temperature at 40 ℃ and the pressure at 0.2MPa, removing thalli, larger proteins and impurity particles, concentrating the solution to the mass concentration of about 55%, and obtaining refined enzymatic conversion solution, namely the raw material; the raw materials comprise about 60% of arbutin, about 30% of hydroquinone and about 10% of isomaltulose by mass percent.
(2) Multi-column batch simulated moving bed separation: according to the difference of affinities of the potassium stationary phase resin to different components, a feed port and a discharge port are configured, and three components of hydroquinone, arbutin and isomaltulose in the raw materials are separated and extracted according to the steps.
The chromatographic column is subjected to heat preservation through dividing wall type heat exchange, and the temperature is 60 ℃; the multi-column intermittent simulated moving bed chromatography uses potassium cation exchange resin as a stationary phase, uses water as an eluent, and has an operating temperature of 60 ℃; the stationary phase is potassium type cation exchange resin.
Before feeding, deionized water at 60 ℃ is fed into the multi-column batch simulated moving bed chromatographic system as an eluent, the flow is kept at 4mL/min, the operation of other pumps is stopped, and the gas remained in the columns is discharged. The separation zone increased the eluent flow to a target value of 6mL/min and the feed flow was 3mL/min.
After all the steps are finished, the position of each feeding and discharging valve is moved forward by one chromatographic column along the liquid flow direction, the initial position of the feeding and discharging valves is recovered after the operation cycle of all the feeding and discharging valves is finished, the steps are repeatedly operated, and isomaltulose components, arbutin components and hydroquinone components are respectively collected.
Repeating the steps, and after 15 times of circulation, the yield of isomaltulose is about 94%, the purity is about 94%, the yield of arbutin is about 91%, and the purity is about 93%; the yield of hydroquinone is about 91% and the purity is about 92%.
The above is only a preferred embodiment of the present invention, and the present invention is not limited to the above examples. It is to be understood that other modifications and variations which may be directly derived or contemplated by those skilled in the art without departing from the spirit and concepts of the present invention are deemed to be included within the scope of the present invention.
Claims (10)
1. A method for separating and extracting arbutin by using simulated moving bed chromatography, which is characterized by comprising the following steps:
(1) Pretreatment of enzymatic conversion solution to obtain a raw material;
(2) Constructing a simulated moving bed chromatographic system;
(3) Simulated moving bed chromatographic separation: introducing the raw material obtained in the step (1) into a simulated moving bed chromatographic system, and separating to prepare arbutin;
in the step (2), the simulated moving bed chromatography comprises a zone I, a zone II, a zone III, a zone IV, a zone V and a zone VI, wherein the zone I, the zone II and the zone III are connected in sequence, the zone IV, the zone V and the zone VI are connected in sequence, and the zone III is disconnected from the zone IV in the initial stage; each zone of the simulated moving bed at least comprises 1 chromatographic column; the chromatograph of the simulated moving bed is divided into a separation area, an enrichment area and an extraction area according to the functional partition.
2. The method according to claim 1, wherein in the step (1), the specific method for pretreatment of the enzymatic conversion solution is as follows: filtering the crude enzymatic conversion solution by a membrane, and concentrating to obtain a raw material; the membrane filtration means filtration by using a nanofiltration membrane, wherein the aperture of the nanofiltration membrane is 50-1000 nm, the filtration pressure is 0.2-0.4 MPa, and the temperature is 30-50 ℃; the concentration refers to concentrating the enzymatic conversion solution filtered by the nanofiltration membrane until the mass concentration is 40% -60%; the raw materials comprise the following components in percentage by mass: the content of arbutin is 40-70%, the content of hydroquinone is 20-40%, and the content of isomaltulose is 10-20%.
3. The method of claim 1, wherein in step (2), the simulated moving bed chromatography system uses potassium or sodium type cation exchange resin as a stationary phase and deionized water as an eluent.
4. A method according to claim 3, wherein the flow rate of the eluent is: before feeding, flowing an eluent into the system, keeping the flow at 4-5 mL/min, stopping the operation of other pumps, and removing the gas remained in the column; the eluent flow rate is increased to a target value of 6-7 mL/min in the separation zone.
5. The method according to claim 1, wherein in step (2), the simulated moving bed chromatography system is operated at a temperature of 60-80 ℃ requiring heat preservation of the chromatography column.
6. The method of claim 1, wherein in step (2), the simulated moving bed chromatography system further comprises a first eluent inlet, a first extract outlet, a feedstock inlet, a first raffinate outlet, a second eluent inlet, a second extract outlet, a first raffinate inlet, a second raffinate outlet;
the area I is positioned between the first eluent inlet and the first extracting solution outlet; the zone II is positioned between the first extracting solution outlet and the raw material inlet; the zone III is located between the feed inlet and the first raffinate outlet; the IV zone is positioned between the second eluent inlet and the second extracting solution outlet; the V zone is positioned between the second extract outlet and the first raffinate inlet; the VI zone is located between the first raffinate inlet and the second raffinate outlet;
the separation zone is located between the feed inlet and the raffinate outlet; the enrichment zone is positioned between the extracting solution outlet and the raw material inlet; the extraction zone is located between the eluent inlet and the extract outlet.
7. The method of claim 6, wherein the first extract comprises hydroquinone; the second extracting solution contains arbutin; the first raffinate contains arbutin and isomaltulose; the second raffinate contains isomaltulose.
8. The method of claim 1, wherein in step (2), the simulated moving bed chromatography system further comprises an eluent inlet valve, an extract valve, a raffinate valve, a feed valve.
9. The method according to claim 1, wherein in step (3), the specific steps of the simulated moving bed chromatographic separation are as follows:
firstly, opening an eluent inlet valve in front of a 1 st chromatographic column in an area I and an eluent inlet valve in front of a 1 st chromatographic column in an area IV and an extraction valve in front of the 1 st chromatographic column in an area II, and then opening a feed valve in front of the 1 st chromatographic column in an area III to input raw material liquid into a system, wherein the feed flow rate of the raw material liquid is 3-5 mL/min, and performing chromatographic separation;
eluent is respectively input into the I zone and the IV zone, and the first raffinate flowing out of the III zone flows to a raffinate inlet between the V zone and the VI zone through a pipeline; the hydroquinone of the first extracting solution flows out between the last 1 chromatographic column of the zone I and the 1 st chromatographic column of the zone II; the tail of the last 1 chromatographic column in the III zone flows out of the first raffinate arbutin and isomaltulose; the tail of the last 1 chromatographic column in the IV zone flows out of the arbutin of the second extracting solution; the tail of the last 1 chromatographic column in the VI zone flows out of the isomaltulose of the second raffinate;
after the eluent inlet, the raw material inlet, the extracting solution outlet and the raffinate outlet run for 1 switching period, the eluent inlet, the raw material inlet, the extracting solution outlet and the raffinate outlet are synchronously switched to the next sequential position along the flowing direction of the eluent, the disconnected position of the chromatographic partition of the simulated moving bed is synchronously switched to the next sequential position along the flowing direction of the eluent, the operation cycle of all the feeding and discharging valves is completed, the initial position of the feeding and discharging valves is recovered, the operation steps are repeated, and the medium retention component arbutin with higher purity can be continuously obtained.
10. The method of claim 9, wherein in step (3), there are two sub-periods within the 1 switching period, the two sub-periods are specifically as follows:
(a) Sub-period one: firstly, separating hydroquinone components from raw materials for 10-16 min;
(b) Sub-period two: and separating the arbutin component from the first raffinate for 8-12 min.
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