CN113698293A - High-purity algae-derived PLA and preparation method thereof - Google Patents
High-purity algae-derived PLA and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a high-purity algae source PLA and a preparation method thereof, which is characterized in that a complete process route from algae oil to a pure PLA product comprises three steps of transesterification, decoloration, purification and the like, wherein the transesterification searches conditions such as catalyst concentration, material ratio, reaction temperature, reaction time and the like, and the conversion rate is improved; the decolorization inspects the types, the dissolving modes, the material ratios and the decolorization time of the adsorbents, so that the loss rate is reduced while the decolorization is ensured; and the purification step adopts a chromatographic separation and purification means, silica gel or silica gel bonding material, organic solvent and water phase are mixed as eluent for separation, and finally the high-purity ethyl ester or methyl ester type PLA product with gas phase purity of more than 98 percent is obtained.
Description
Technical Field
The invention relates to the technical field of transesterification, decoloration and purification preparation of algae-derived PLA.
Background
Unsaturated fatty acid has important prevention, health care and treatment effects in antioxidation, cardiovascular and cerebrovascular maintenance, neural development and repair, metabolic syndrome regulation and control and the like, but the function and safety of the unsaturated fatty acid are greatly influenced by purity and impurity content. The algae-derived fatty acid is an important source of sustainable unsaturated fatty acid, but the composition is complex; compared with fish oil fatty acid, the fatty acid extracted from the marine microalgae has no peculiar smell and does not contain cholesterol, the content of unsaturated fatty acid in some algae is far higher than that of the fish oil, and the contained species are pure and convenient to analyze. Lipid resources rich in unsaturated fatty acid are extracted from the algae, and a foundation is laid for deep processing of the marine microalgae; how to solve the problem of efficient directional preparation of the target unsaturated fatty acid is the key for promoting the industrialization of the unsaturated fatty acid of the microalgae.
The content of palmitoleic acid (C16:1 delta 9) in the yellow silk algae oil accounts for about 50% of the total fatty acid, which is far higher than that of other common microalgae (Wang et al, 2016). Palmitoleic acid (C16:1 delta 9) belongs to omega-7 monounsaturated fatty acid, has the effects of promoting triglyceride reduction and benefiting cardiovascular system health like omega-3 fatty acid, is also considered as a novel hormone, can improve the sensitivity of a human body to insulin, and has important effects on the prevention, health care and treatment of type II diabetes. Researches show that the xanthoceras fulva can utilize organic carbon sources, particularly glucose, to perform high-density heterotrophic growth (Wang et al, 2017; Zhou et al, 2017), and a batch type fed-batch fermentation process of the xanthoceras fulva is established by optimizing organic carbon, nitrogen sources, phosphorus sources and inorganic nutrient salts in a culture medium, so that the final biomass can reach 40g/L, and the oil content can reach about 45% of the dry weight after induction. The technology lays a foundation for establishing a process for producing omega-7 fatty acid and co-producing biodiesel by utilizing heterotrophic fermentation of the chrysophyceae.
The lipid mixture extracted from algae without any purification treatment is called algae oil, in which palmitoleic acid is of carboxylic acid type and which contains a large amount of pigments, mainly chlorophyll. The presence of chlorophyll gives algal oil a brownish black or dark green color; on the basis of an established high-efficiency unsaturated fatty acid preparation chromatographic separation system and the directional production of the algae-derived unsaturated fatty acid, the research on the high-efficiency preparation technology of the practical microalgae unsaturated fatty acid is carried out by taking omega-7 palmitoleic acid in the xanthoceras fulva as an object according to the target requirements of product purity and impurity control, and a linkage and scale amplification technical system for supplying the algae-derived unsaturated fatty acid resource to the whole high-efficiency preparation chromatographic purification process is established to realize the high-efficiency production preparation of the industrially-amplified high-purity algae-derived omega-7 palmitoleic acid.
Disclosure of Invention
The invention relates to a method for performing transesterification, decoloration and two-step chromatographic separation and purification on crude algae oil, which comprises the following steps:
1) algal oil transesterification: weighing a proper amount of algae oil original sample, and adding the algae oil original sample into a prepared 1-10% sulfuric acid-ethanol or methanol solution, wherein the volume ratio of the mass of the algae oil to ethanol or methanol to sulfuric acid is 1: 5-1: 100, heating in an oil bath until the solution boils slightly, wherein the temperature is 60-80 ℃, and heating and refluxing for 0.5-5 h; after the reaction is finished, cooling the solution, adding a proper amount of pure water and n-hexane for extraction, collecting the n-hexane layer, and carrying out reduced pressure concentration to obtain a dark green or tan ethyl ester or methyl ester type PLA crude product;
2) and (3) decoloring: weighing a proper amount of ethyl ester or methyl ester type PLA crude product, dissolving in one or more of methanol, ethanol, n-hexane, acetone, isopropanol and acetonitrile, mixing with one or more of decolorant, silica gel, active carbon, carclazyte, attapulgite, graphitized carbon, resin and the like, and stirring, wherein the mass ratio of the decolorant to the sample is 1: 0.5-1: 10, the decoloring time is as follows: 0.1-1 h; after the reaction is finished, filtering the sample liquid, and removing the organic solvent by rotary evaporation to obtain a light yellow ethyl ester or methyl ester type PLA crude product;
3) chromatographic one-dimensional separation and purification: weighing a proper amount of the ethyl ester or methyl ester type PLA crude product obtained in the step 2), and preparing the ethyl ester or methyl ester type PLA crude product into a product with the concentration of 10-800 mg/mL by using initial flow matching; hydrophobic filler (particle diameter of 5-100 μm, pore diameter of
(ANG) and a specific surface area of 100 to 500m2Per g, the mass content of carbon in the filler is 5-35%, and the bonded alkyl stationary phase has one or more of 1-30 carbon atoms in normal chain alkyl) for separation; the eluent is water solution and methanol, ethanol, acetonitrile, acetone,the volume ratio of the water phase to the organic phase in the mixed solution of isopropanol in the eluent is usually 50-100% of the volume of the organic phase; the flow rate and flow velocity of the eluent are 0.2mm/s-2mm/s, the temperature is 10-60 ℃, and the ultraviolet detection wavelength is 200nm-220 nm; collecting target peak fraction liquid, and performing gas phase detection to obtain a sample obtained by one-dimensional separation;
4) and (3) replacing the sample obtained by the one-dimensional separation in the step 3) with the sample obtained by the one-dimensional separation in the step 3) after the raw material is decolorized, and repeating the step 3) to complete the second column chromatographic separation to obtain a sample obtained by the two-dimensional separation, namely a product.
The innovation points and advantages of the invention are as follows:
1. the complete process route from the algae oil to the pure ethyl ester or methyl ester type PLA product comprises three steps of transesterification, decoloration, purification and the like, and a product with the gas phase purity of more than 98 percent can be obtained;
2. the sample loading amount is large, and the industrial production is easy to realize;
3. the process flow is simple, the repeatability is good, the operation is simple and controllable, the automation is easy to realize, and the process is stable;
4. the filler in the chromatographic column can be repeatedly used, so that the cost is saved;
drawings
FIG. 1 spectrum of one-dimensional purification preparation in example 1
FIG. 2 two-dimensional purification preparation spectrum in example 1
FIG. 3 gas phase analysis spectra of PLA standards used in examples 1-4
FIG. 4 gas phase analysis of one-dimensional PLA product in example 1
FIG. 5 gas phase analysis of two-dimensional PLA product in example 1
As can be seen from FIGS. 1 to 5 (except FIG. 3), the purity of the sample preparation process and the sample gas chromatography changes from low to high.
Detailed Description
The present invention will now be further described with reference to examples. The examples are given solely for the purpose of illustration and are not intended to be limiting.
The conditions of the gas chromatography carried out in the examples used below were as follows:
a chromatographic column: DB-5: 0.25mm 0.25 μm 30 m;
temperature:
sample introduction end: 320 deg.C
Detecting end temperature: 350 deg.C
A chromatographic column section: please refer to the following temperature schedule
Gas: nitrogen gas
Flow rate: 1.3 mL/min;
sample introduction volume: 1 mu L of the solution;
example 1
1) Algal oil transesterification: weighing 40g of yellow silk algae oil, adding the original yellow silk algae oil into 1000mL of prepared 3% sulfuric acid (with mass concentration of 98%) -ethanol solution, heating in an oil bath until the solution boils slightly, and heating and refluxing for 2 hours at the temperature of 75 ℃; after the reaction is finished, cooling the solution, adding 100mL of pure water, extracting with n-hexane for three times, and extracting with 100mL of n-hexane each time; collecting the n-hexane layer, and concentrating under reduced pressure at 30 deg.C to obtain 39.26g of dark green or brown ethyl ester type PLA oily crude product with a mass recovery rate of 98.15%; 2) and (3) decoloring: weighing 35g of the ethyl ester type PLA crude product in the step 1), dissolving the ethyl ester type PLA crude product in 350mL of methanol solution, adding 7g of silica gel, shaking, standing for 0.5h, filtering the sample solution, and removing the organic solvent by rotary evaporation at 60 ℃ to obtain 33.25g of light yellow ethyl ester type PLA oily crude product, wherein the mass recovery rate is 95%, and the gas chromatography purity of the sample is 37.12%;
3) chromatographic one-dimensional separation and purification: weighing 30g of the ethyl ester type PLA crude product obtained in the step 2), adding an initial mobile phase (elution solvent) to dissolve to 60mL, and completely injecting samples, wherein the sample loading amount (the mass ratio of the raw material to the filler) is 10%; using octadecyl filler bonded on the surface of silica gel (column specification 50X 250mm (diameter and height), particle diameter 15 μm, pore diameter
(angstrom), a carbon content of 15% by mass in the filler, a specific surface area of 300m2(g, filler mass 300g), column temperature 30 ℃ and flow rate0.7 mm/s; eluting solvent is methanol and water, the volume ratio of the methanol to the water phase is 90/10, detecting wavelength is 210nm by an ultraviolet detector, separating target peak distillate, and performing gas phase detection to obtain 14.83g of one-dimensional PLA oily product with the gas chromatography purity of 75.08%, wherein the recovery rate of the target peak is 90%;
4) chromatographic two-dimensional separation and purification: 10g of the obtained sample in the step 3) is added with an initial mobile phase (elution solvent) and dissolved to 20mL, and the loading amount (the mass ratio of the raw material to the filler) is 3.3%; using octadecyl filler (column size 50X 250mm (diameter and height), particle diameter 15 μm, pore diameter) bonded on the surface of silica gel
(angstrom), a carbon content of 15% by mass in the filler, a specific surface area of 300m2The mass of the filler is 300g), the column temperature is 30 ℃, and the flow rate is 0.7 mm/s; eluting solvent is methanol and water, the volume ratio of the methanol to the water phase is 95/5, detecting wavelength is 210nm by an ultraviolet detector, separating target peak distillate, and performing gas phase detection to obtain 9.36g of a two-dimensional PLA oily product with gas chromatography purity of 98.5%, wherein the recovery rate of the target peak is 92%;
example 2
1) Algal oil transesterification: weighing 40g of yellow silk algae oil, adding the original yellow silk algae oil into 1200mL of prepared 3% sulfuric acid (with mass concentration of 98%) -methanol solution, heating the solution in an oil bath until the solution boils slightly, and heating and refluxing the solution for 2 hours at the temperature of 75 ℃; after the reaction is finished, cooling the solution, adding 100mL of pure water, extracting with n-hexane for three times, and extracting each time with 100 mL; collecting the n-hexane layer, and concentrating under reduced pressure at 30 deg.C to obtain 39.74g dark green or brown methyl ester type PLA oily crude product with a mass recovery rate of 99.35%;
2) and (3) decoloring: weighing 35g of the methyl ester type PLA crude product in the step 1), dissolving the 35g of the methyl ester type PLA crude product in 35mL of methanol solution, adding 7g of argil, shaking, standing for 0.5h, filtering the sample solution, and removing the organic solvent by rotary evaporation at 60 ℃ to obtain 34.3g of light yellow methyl ester type PLA oily crude product, wherein the mass recovery rate is 98%, and the purity of the sample gas chromatography is 37.46%;
3) chromatographic one-dimensional separation and purification: weighing 45g of the methyl ester type PLA crude product obtained in the step 2), adding an initial mobile phase (elution solvent) to dissolve to 90mL, completely injecting samples,the loading amount (mass ratio of raw materials to the filler) is 15%; using silica gel surface bonded octadecyl filler (column size 50X 250mm (diameter and height), particle diameter 12 μm, pore diameter
(angstrom), a carbon content in the filler of 18% by mass and a specific surface area of 300m2The mass of the filler is 300g), the column temperature is 30 ℃, and the flow rate is 0.7 mm/s; eluting solvent is methanol and water, the volume ratio of the methanol to the water phase is 90/10, detecting wavelength is 210nm by an ultraviolet detector, separating target peak distillate, and performing gas phase detection to obtain 20.95g of one-dimensional PLA oily product with the gas chromatography purity of 70.02%, and the target peak recovery rate is 87%;
4) chromatographic two-dimensional separation and purification: adding 20g of the obtained sample in the step 3) into the initial mobile phase to dissolve the sample to 40mL, wherein the loading amount (mass ratio of the raw material to the filler) is 6.7%; using silica gel surface bonded octadecyl filler (column size 50X 250mm (diameter and height), particle diameter 12 μm, pore diameter
(angstrom), a carbon content in the filler of 18% by mass and a specific surface area of 300m2The mass of the filler is 300g), the column temperature is 30 ℃, and the flow rate is 0.5 mm/s; eluting solvent is methanol and water, the volume ratio of the methanol to the water phase is 95/5, detecting wavelength is 210nm by an ultraviolet detector, separating target peak distillate, and performing gas phase detection to obtain 13.70g of a two-dimensional PLA oily product with gas chromatography purity of 98.6%, wherein the recovery rate of the target peak is 92%;
example 3
1) Algal oil transesterification: weighing 60g of yellow silk algae oil, adding the original yellow silk algae oil into 1200mL of prepared 5% sulfuric acid (with mass concentration of 98%) -ethanol solution, heating in an oil bath until the solution boils slightly, and heating and refluxing for 2 hours at the temperature of 75 ℃; after the reaction is finished, cooling the solution, adding 100mL of pure water, extracting with n-hexane for three times, and extracting each time with 100 mL; collecting the n-hexane layer, and concentrating under reduced pressure at 30 deg.C to obtain 58.81g of dark green or brown ethyl ester type PLA oily crude product with a mass recovery rate of 98.01%;
2) and (3) decoloring: weighing 50g of the ethyl ester type PLA crude product in the step 1), dissolving the ethyl ester type PLA crude product in 100mL of n-hexane solution, adding 10g of attapulgite, shaking, standing for 0.5h, filtering the sample solution, and removing the organic solvent by rotary evaporation at 60 ℃ to obtain 49g of light yellow ethyl ester type PLA oily crude product, wherein the mass recovery rate is 98%, and the gas chromatography purity of the sample is 37.43%;
3) chromatographic one-dimensional separation and purification: weighing 45g of the ethyl ester type PLA crude product obtained in the step 2), adding an initial mobile phase (elution solvent) to dissolve to 90mL, and completely injecting samples, wherein the sample loading amount (the mass ratio of the raw material to the filler) is 15%; silica gel surface-bonded hexadecyl filler (column size 50X 250mm (diameter and height), particle size 12 μm, pore diameter
(angstrom), a carbon content in the filler of 18% by mass and a specific surface area of 300m2The mass of the filler is 300g), the column temperature is 30 ℃, and the flow rate is 0.7 mm/s; eluting solvent is ethanol and water, the volume ratio of the ethanol to the water phase is 90/10, detecting wavelength is 210nm by an ultraviolet detector, separating target peak distillate, and performing gas phase detection to obtain 19.75g of one-dimensional PLA oily product with gas chromatography purity of 75.12%, wherein the target peak recovery rate is 88%;
4) chromatographic two-dimensional separation and purification: adding 20g of the obtained sample in the step 3) into the initial mobile phase to dissolve the sample to 40mL, wherein the loading amount (mass ratio of the raw material to the filler) is 6.7%; silica gel surface-bonded hexadecyl filler (column size 50X 250mm (diameter and height), particle size 12 μm, pore diameter
(angstrom), a carbon content in the filler of 18% by mass and a specific surface area of 300m2The mass of the filler is 300g), the column temperature is 30 ℃, and the flow rate is 0.5 mm/s; eluting solvent is ethanol and water, the volume ratio of the ethanol to the water phase is 95/5, detecting wavelength is 210nm by an ultraviolet detector, separating target peak distillate, and performing gas phase detection to obtain 13.64g of two-dimensional PLA oily product with gas chromatography purity of 99.1%, wherein the recovery rate of the target peak is 90%;
example 4
1) Algal oil transesterification: weighing 60g of yellow silk algae oil, adding the original yellow silk algae oil into 1800mL of prepared 5% sulfuric acid (with mass concentration of 98%) -methanol solution, heating the solution in an oil bath until the solution boils slightly, and heating and refluxing the solution for 2 hours at the temperature of 75 ℃; after the reaction is finished, cooling the solution, adding 100mL of pure water, extracting with n-hexane for three times, and extracting each time with 100 mL; collecting the n-hexane layer, and concentrating under reduced pressure at 30 deg.C to obtain 58.81g dark green or brown methyl ester type PLA oily crude product with a mass recovery rate of 98.01%;
2) and (3) decoloring: weighing 50g of the methyl ester type PLA crude product in the step 1), dissolving the methyl ester type PLA crude product in 100mL of ethanol solution, adding 10g of argil, shaking, standing for 0.5h, filtering the sample solution, and removing the organic solvent by rotary evaporation at 60 ℃ to obtain 47.5g of light yellow methyl ester type PLA oily crude product, wherein the mass recovery rate is 95%, and the purity of the sample gas chromatography is 37.26%;
3) chromatographic one-dimensional separation and purification: weighing 15g of the methyl ester type PLA crude product obtained in the step 2), adding an initial mobile phase (elution solvent) to dissolve to 30mL, and completely injecting samples with the sample loading amount (mass ratio of raw materials to filler) of 5%; using silica gel surface bonded octadecyl filler (column size 50X 250mm (diameter and height), particle diameter 12 μm, pore diameter
(angstrom), a carbon content in the filler of 19% by mass and a specific surface area of 300m2The mass of the filler is 300g), the column temperature is 30 ℃, and the flow rate is 0.6 mm/s; eluting solvent is ethanol and water, the volume ratio of ethanol to water phase is 90/10, detecting wavelength is 210nm by an ultraviolet detector, separating target peak distillate, and performing gas phase detection to obtain 6.38g of one-dimensional PLA oily product with gas chromatography purity of 79.25%, wherein the target peak recovery rate is 90.5%;
4) chromatographic two-dimensional separation and purification: adding the initial mobile phase into 6g of the obtained sample in the step 3) to dissolve to 12mL, wherein the loading amount (mass ratio of the raw material to the filler) is 2%; using silica gel surface bonded octadecyl filler (column size 50X 250mm (diameter and height), particle diameter 12 μm, pore diameter
(angstrom), a carbon content of 192% by mass in the filler, and a specific surface area of 300m2The mass of the filler is 300g), the column temperature is 30 ℃, and the flow rate is 0.5 mm/s; eluting with ethanol and water at a volume ratio of ethanol to water of 95/5, detecting with ultraviolet detectorThe length is 210nm, the target peak fraction is separated and gas phase detection is carried out, 4.54g of two-dimensional PLA oily product with the gas chromatography purity of 99.5% is obtained, and the target peak recovery rate is 95%.
Claims (10)
1. A high purity palmitoleic acid characterized by: the product PLA (palmitoleic acid C16:1, n-7cis) is one or two of omega-7 hexadecenoic acid ethyl ester or omega-7 hexadecenoic acid methyl ester with the normalized purity of the gas chromatography peak area being more than or equal to 98 percent, and the structural formula of the omega-7 hexadecenoic acid ethyl ester or the omega-7 hexadecenoic acid methyl ester is as follows:
2. a process for the production of palmitoleic acid as claimed in claim 1, characterized by: the oil of the yellow silk algae is prepared by three steps of transesterification, adsorption decoloration and column chromatography separation and purification;
the transesterification step is as follows: mixing algae oil with ethanol and/or methanol and sulfuric acid, and heating in oil bath for reflux reaction; and after the transesterification reaction is finished, cooling to 20-40 ℃, adding deionized water, extracting with n-hexane, collecting the n-hexane layer, and performing reduced pressure concentration to remove the n-hexane to obtain the transesterified algae oil.
3. The method of claim 2, wherein: the volume ratio (g/ml) of the mass of the transesterified algae oil to the ethanol and/or methanol and the sulfuric acid is 1: 5-1: 100 (preferably 1: 10-1: 30);
the volume ratio of concentrated sulfuric acid (mass concentration 95-98%) as a catalyst required by transesterification to ethanol and/or methanol is as follows: 1% to 10% (preferably 3% to 5%).
4. The method of claim 2, wherein: the reaction time of transesterification is 0.5-5 h (preferably 2-4 h); the reaction temperature of transesterification is 60-85 ℃ (preferably 70-75 ℃).
5. The method of claim 2, wherein: the usage amounts of the deionized water and the normal hexane are respectively 2-20% (preferably 3-10%) of the volume of the system after the transesterification reaction is finished.
6. The method of claim 2, wherein:
the decoloring step is that the sample after transesterification is dissolved by an organic solvent and then is mixed with a decoloring agent, and the mixture is stood, filtered and decompressed and concentrated to remove the organic solvent;
the decolorizing agent is one or more of silica gel, activated carbon, carclazyte, attapulgite, graphitized carbon, macroporous adsorption resin and other materials;
the mass ratio of the decoloring agent to the sample is 1: 0.5-1: 10 (preferably 1: 2 to 1: 5);
standing and decoloring time is as follows: 0.1 to 1 hour (preferably 0.2 to 0.5 hour);
when in decoloring, the organic solvent for dissolving the sample is one or more of methanol, ethanol, normal hexane, acetone, isopropanol and acetonitrile;
the concentration of the sample after the organic solvent is dissolved is 10-500 mg/mL (preferably 200-500 mg/mL).
7. The method of claim 2, wherein:
the column chromatography separation needs to be carried out twice, and the two purification processes are as follows:
1) sample dissolving: dissolving the decolored sample by using an organic solvent or a mixed solution of the organic solvent and an aqueous solution, and filtering after complete dissolution to obtain a dissolved solution; the volume concentration of the organic solvent in the solution for dissolving is between 50 and 100 percent (preferably between 70 and 95 percent);
2) preparing a mobile phase: the mobile phase adopts the organic solvent or the mixed solution of the organic solvent and the aqueous solution in the step 1), and the volume concentration of the organic solvent in the mobile phase is between 50 and 100 percent (preferably between 70 and 95 percent);
3) and (3) eluting on a column: pumping the dissolving solution into a preparation column filled with the hydrophobic filler, eluting by using the mobile phase prepared in the step 2), collecting a PLA sample, and sampling for gas phase analysis;
4) concentration: concentrating the collected target components to complete the first column chromatographic separation to obtain a sample obtained by one-dimensional separation;
5) and (3) replacing the sample obtained by the one-dimensional separation in the step 4) with the sample obtained by the decoloration of the raw material in the step 1) to repeat the processes in the steps 1) to 4), and completing the second column chromatographic separation to obtain a sample obtained by the two-dimensional separation, namely a product.
8. The method of claim 7, wherein:
the organic solvent in the step 1) is one or more of methanol, ethanol, acetonitrile, acetone, isopropanol and the like;
step 1), dissolving the sample solution by using an initial mobile phase (namely the adopted mobile phase), wherein the concentration of the sample is between 10 and 800mg/mL (preferably 300 to 500mg/mL), and the amount of the sample adsorbed on the upper column is 0.1 to 20 percent (preferably 5 to 15 percent) of the weight of the filler.
9. The method of claim 7, wherein:
step 3) the hydrophobic filler is a silica gel surface bonded alkyl stationary phase, the mass content of carbon in the filler is 5-35% (preferably 10-20%), and the alkyl is one or more of normal chain alkyl with 1-30 carbon atoms;
the hydrophobic filler in the step 3) is characterized in that the particle diameter is 5-100 mu m (preferably 10-60 mu m), and the pore diameter is
(Angstrom) (preferably
(angstrom)) has a specific surface area of 100 to 500m2(preferably 200 to 400 m)/g2/g)。
10. The method of claim 7, wherein:
step 3), the inner diameter of a chromatogram used by the high-efficiency preparative chromatogram is 20 mm-1600 mm, and the height of a chromatographic column bed is 100 mm-1000 mm;
and 3) in the high-efficiency preparative chromatography purification, the flow velocity of a flowing phase is 0.2-2 mm/s (preferably 0.3-1 mm/s), the temperature is 10-60 ℃ (preferably 20-40 ℃), an ultraviolet detector is adopted for detection in the elution process, and the ultraviolet detection wavelength is 200-220 nm.
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