CN113292713A - Molecular weight grading and purifying method of polyhydroxyalkanoate - Google Patents
Molecular weight grading and purifying method of polyhydroxyalkanoate Download PDFInfo
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- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/88—Post-polymerisation treatment
- C08G63/90—Purification; Drying
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J11/00—Recovery or working-up of waste materials
- C08J11/04—Recovery or working-up of waste materials of polymers
- C08J11/10—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation
- C08J11/14—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation by treatment with steam or water
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J11/00—Recovery or working-up of waste materials
- C08J11/04—Recovery or working-up of waste materials of polymers
- C08J11/10—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation
- C08J11/16—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation by treatment with inorganic material
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2367/00—Characterised by the use of polyesters obtained by reactions forming a carboxylic ester link in the main chain; Derivatives of such polymers
- C08J2367/04—Polyesters derived from hydroxy carboxylic acids, e.g. lactones
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- Y02W30/62—Plastics recycling; Rubber recycling
Abstract
The invention relates to the field of biomedicine, and more specifically relates to a method for classifying and purifying the molecular weight of polyhydroxyalkanoate, which comprises the following steps: s1, dissolving the polyhydroxy fatty acid ester raw material in a good solvent, and adding a catalyst for heating degradation; s2, cooling the solution after the degradation by heating in the step S1 to room temperature, mixing the solution with a poor solvent, precipitating and separating out the degradation product, and collecting the degradation product after the precipitation; s3, after the degradation product collected in the step S2 is centrifuged, the degradation product is washed and dried, and thus the polyhydroxy fatty acid ester is subjected to molecular weight classification and purification. The method has simple steps and convenient operation, and can carry out molecular weight classification while purifying the polyhydroxyalkanoate, and the polyhydroxyalkanoate obtained by the classification and purification of the method has high purity, uniform and accurate molecular weight, narrower molecular weight distribution and higher active terminal.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a molecular weight grading and purifying method of polyhydroxyalkanoate.
Background
Biopolymer materials obtained by biofermentation are one of the most rapidly developing biomedical friendly materials in recent years. Among the biomaterials, Polyhydroxyalkanoate (PHA) is especially excellent in performance, and a nerve conduit made of the PHA is reported to be a material with the auxiliary functions of promoting cartilage material regeneration and esophagus regeneration, so that the PHA proves good biocompatibility and excellent physicochemical and mechanical properties, poly-4-hydroxybutyrate (P4HB) in the PHA family is used as a patch for repairing abdominal wall defects of human bodies, the patch has better biocompatibility compared with a traditional polypropylene (PP) patch, but has the defect of high cost, and no related products exist in China, the main reasons are that the preparation of medical grade polyester is difficult, namely the molecular weight requirement is accurate, the purity requirement is high, and poly-3-hydroxybutyrate-co-4-hydroxybutyrate (P34HB) as a fourth-generation PHA product is the most excellent in performance in the PHA family, 3-hydroxybutyric acid is contained in the degradation product of the compound, so that the compound is helpful for repairing biological tissues. And the proportion of the 3-hydroxybutyric acid and the 4-hydroxybutyric acid is changed, so that the physical, chemical and mechanical properties of the biological resin can be greatly optimized.
In recent years, the basic research on Polyhydroxyalkanoate (PHA) in China has been completed basically by obtaining PHA through biological fermentation, collecting and purifying the PHA and processing PHA devices, for example, the Chen nationality strong team of the Qinghua university in China has strictly screened and successfully obtained two kinds of salt-tolerant bacteria, the large-scale production of a series of PHA is realized through microbial fermentation, medical PHA is obtained through improving a purification method, if the substitution of the traditional polyolefin plastics is realized, the white pollution caused by chemical plastics can be effectively solved, however, as the biological fermentation has a plurality of defects relative to the chemical process, the basic chemical parameters such as the product molecular weight and the like are difficult to control, the subsequent purification process often contains residues of cell membranes and cytoplasm, and also contains certain endotoxin, the obtained PHA raw material is not pure enough, and industrialization is difficult to realize. The main problem is therefore that most PHA feedstocks are of low purity and of broad molecular weight distribution, which severely limits their refinement and functional applications and hence lacks further fine processing of the PHA feedstock.
In order to solve the problem of low PHA purity, the Chinese patent "purification method of polyhydroxyalkanoate" (application No. 201910001260.3, published: 2019.03.26) discloses a purification method of polyhydroxyalkanoate, which comprises the following steps: (1) dissolving the PHA crude product in an organic solvent, heating, stirring and cooling simultaneously, and then carrying out centrifugal separation; the organic solvent is mutually soluble with water; (2) adding water into the separated solution, centrifuging and collecting precipitate; (3) the purification of the polyhydroxyalkanoate is realized by cleaning the precipitate with water, and the purity of the PHA can be improved by the method, but the purified PHA has a wider molecular weight distribution, and the PHA cannot be refined and functionalized. However, there are not many patents related to the physicochemical properties of PHA, such as molecular weight and distribution control.
The technical difficulty in obtaining high-purity narrow molecular weight distribution polyester is that the chemical composition of the polyester obtained by biological fermentation is complex, the molecular weight distribution is wide, and the purification difficulty is high, wherein the biggest challenge is how to controllably obtain PHA molecules with different molecular weights and molecular weight distributions. Therefore, it is desirable to provide a method for molecular weight fractionation and purification of PHA simultaneously, so as to achieve integration of molecular weight regulation and purification.
Disclosure of Invention
The present invention is directed to overcoming at least one of the above-mentioned drawbacks of the prior art and providing a method for classifying and purifying polyhydroxyalkanoates by molecular weight, which has simple steps and convenient operation, and can classify the polyhydroxyalkanoates by molecular weight while purifying it, and polyhydroxyalkanoates obtained by the method have high purity, uniform and accurate molecular weight, narrow molecular weight distribution, and higher active terminals.
The invention adopts the technical scheme that the method for grading and purifying the molecular weight of the polyhydroxyalkanoate comprises the following steps:
s1, dissolving the polyhydroxy fatty acid ester raw material in a good solvent, and adding a catalyst for heating degradation;
s2, cooling the solution after the degradation by heating in the step S1 to room temperature, mixing the solution with a poor solvent, precipitating and separating out the degradation product, and collecting the degradation product after the precipitation;
s3, after the degradation product collected in the step S2 is centrifuged, the degradation product is washed and dried, and thus the polyhydroxy fatty acid ester is subjected to molecular weight classification and purification.
In the technical scheme, a settling method is adopted to carry out settling purification on a polyhydroxyalkanoate raw material, the polyhydroxyalkanoate raw material is dissolved in a good solvent, the dissolution and the degradation are promoted by heating, the molecular weight of polyhydroxyalkanoate with larger molecular weight and longer chain length is reduced after degradation, so that the molecular weight distribution of polyhydroxyalkanoate obtained after fractional purification is narrower, the dispersity is smaller, the addition of a catalyst can promote the degradation reaction, the ester bond of polyhydroxyalkanoate is hydrolyzed, the degradation reaction time is greatly shortened, the fractional regulation and purification of the molecular weight of the polyhydroxyalkanoate raw material can be realized in a shorter time, the solution after the heating degradation is mixed with a poor solution, and the polyhydroxyalkanoate is separated out by utilizing the lower solubility between the polyhydroxyalkanoate and the poor solvent, and other components such as cell debris, The pigment or impure polyhydroxyalkanoate is dissolved in a poor solvent, and precipitated polyhydroxyalkanoate can be collected by centrifugation, thereby increasing the collection speed.
Preferably, in step S2, the degraded solution heated in step S1 is cooled to room temperature of 30 ℃.
Preferably, the precipitation time of the degradation product is 1-6 h.
Furthermore, the polyhydroxy fatty acid ester raw material is poly 3-hydroxybutyrate-co-4 hydroxybutyrate, the ratio of a 3-hydroxybutyrate monomer and a 4-hydroxybutyrate monomer of the poly 3-hydroxybutyrate-co-4 hydroxybutyrate is (1-10) - (10-1), and the purity of the poly 3-hydroxybutyrate-co-4 hydroxybutyrate is more than 85%.
Furthermore, the number average and weight average relative molecular weight of the polyhydroxy fatty acid ester raw material is 20-80 ten thousand, and the molecular weight dispersity is 1.5-2.5.
Preferably, the purity of the polyhydroxyalkanoate raw material is characterized by liquid permeation gel chromatography (GPC), hydrogen nuclear magnetic resonance spectroscopy (H-NMR) and Thermogravimetry (TGA) to screen polyhydroxyalkanoates having a purity of 85% or more.
In the technical scheme, the ratio of the 3-hydroxybutyrate monomer and the 4-hydroxybutyrate monomer of the poly-3-hydroxybutyrate-co-4-hydroxybutyrate has a large influence on the molecular weight and the dispersity of the poly-3-hydroxybutyrate-co-4-hydroxybutyrate, so that the monomer ratio, the weight average relative molecular weight and the molecular weight dispersity of the poly-3-hydroxybutyrate-co-4-hydroxybutyrate are controlled before molecular weight classification and purification so as to ensure the subsequent molecular weight regulation and control effect, the purity of the poly-3-hydroxybutyrate-co-4-hydroxybutyrate is characterized and controlled before molecular weight classification and purification, and the influence on the yield and the quality of products due to the existence of excessive impurities can be avoided.
Further, in step S1, the good solvent is one or more of N, N-dimethylformamide, N-dimethylacetamide, chloroform, dichloromethane or dioxane.
In the technical scheme, a solvent with a good dissolving effect on polyhydroxyalkanoate is selected as a good solvent, and since polyhydroxyalkanoate is a polyester type macromolecule and hydrogen bond crystallization is easily formed between molecules, for fully dissolved PHA, a solvent with high polarity, high solubility, high boiling point and high vapor pressure is selected as the good solvent, the dissolving time is regulated and controlled to enable PHA to be fully dissolved in the good solvent, so that a macromolecular chain is fully stretched in the solvent, further degradation reaction is carried out to carry out molecular weight grading and impurity dissolution, and the selected good solvent has high solubility on polyhydroxyalkanoate, so that batch amplification experiments and production are facilitated.
Further, in step S1, the catalyst is water, a weak base solution, or a weak acid solution.
Preferably, in step S1, the catalyst is water, a saturated sodium carbonate solution, a saturated sodium bicarbonate solution, an acetic acid solution, or other weak acid base solution.
More preferably, in step S1, the catalyst is water.
Further, in step S1, the mass ratio of the catalyst to the polyhydroxyalkanoate raw material is 1 (40-100).
Further, in step S1, the time for dissolving the polyhydroxyalkanoate and the good solvent is 2-48 h, and the polyhydroxyalkanoate and the good solvent are heated and degraded at 70-140 ℃ for 1-24 h after the catalyst is added.
Preferably, in step S1, the catalyst is added to the solution of the polyhydroxyalkanoate raw material and the good solvent 1 to 10 times, and the time interval between each addition of the catalyst is 10 to 30 min.
In the technical scheme, the polyhydroxy fatty acid ester raw material can be fully dissolved in the good solvent by controlling the mass ratio of the polyhydroxy fatty acid ester raw material to the good solvent, so that the molecular chain stretching is facilitated, and the higher yield can be obtained.
In the technical scheme, the reaction temperature is controlled to be 10-60 ℃ below the boiling point of the good solvent, so that the degradation effect is prevented from being influenced by excessive volatilization of the good solvent; the adding mode of the catalyst has great influence on the degradation reaction of the polyhydroxyalkanoate, and the catalyst is added for a plurality of times at intervals, so that the degradation reaction is promoted to be continuously carried out.
Preferably, when the reaction vessel is heated and degraded, nitrogen gas is filled above the reaction vessel for protection, so that the occurrence of side reactions is reduced, and the purity of the product is ensured.
Further, in step S2, the poor solvent is one or more of pure water, methanol, or ethanol.
Furthermore, the mass ratio of the polyhydroxy fatty acid ester raw material to the good solvent is 1 (20-100).
Further, the volume ratio of the good solvent to the poor solvent is 1 (30-200), and/or the mass ratio of the good solvent to the poor solvent is 1 (10-200).
In the technical scheme, the solvent with higher solubility with the polyhydroxyalkanoate is selected as the good solvent, so that the polyhydroxyalkanoate can be completely dissolved in the good solvent to carry out degradation reaction, thereby carrying out molecular weight classification, being beneficial to subsequent purification, having higher solubility with the polyhydroxyalkanoate, being more beneficial to batch amplification experiment and production, and determining the type of the good solvent to be selected according to actual needs; the solvent with lower solubility with the polyhydroxyalkanoate is selected as a poor solvent (precipitator) to precipitate the degraded polyhydroxyalkanoate, and different poor solvents have different solubilities on the polyhydroxyalkanoate and impurities, which affects the yield and purity of the final product, so that the type of the poor solvent and the ratio of the good solvent to the poor solvent can be determined according to different requirements. For example, the use of more ethanol may provide better removal of cellular endotoxins.
Further, in step S3, the rotation speed of the centrifuge during the centrifugal separation is 500 to 10000rad · S-1And the centrifugation time is 2-30 min.
Preferably, in the centrifugal process, after washing for 3-10 times by using a poor solvent, adding a proper amount of water, shaking, stirring and mixing uniformly.
Preferably, the time for the first precipitation of the degradation product is 1-6 hours, and the time for each precipitation is 0.5-5 hours.
Preferably, the degradation product is finally washed by pure water for 5-10 times.
Preferably, the degradation product is dried by a freeze-drying or rotary evaporation method after being washed, so as to obtain a powdery product which is subjected to molecular weight classification and purification.
More preferably, the degradation product is lyophilized for 12-72 hours at-20 ℃ and under the vacuum degree of less than 100Pa, so as to ensure the drying of the sample.
More preferably, the degradation product is subjected to water bath at 50-90 ℃ and rotary evaporation at the rotating speed of 20-100 rad.s < -1 >.
Compared with the prior art, the invention has the beneficial effects that:
the method has the advantages of simple steps, convenient operation, safe reagent sources and environment-friendly and safe process, the polyhydroxy fatty acid ester is purified, the catalyst is added to promote the degradation reaction of the polyhydroxy fatty acid ester with larger molecular weight, and the number average molecular weight and the weight average molecular weight of the poly-3-hydroxybutyrate-co-4 hydroxybutyrate obtained by fractional purification by the method are in the range of 1-10 ten thousand, namely 1-20 g.mol-1And the dispersity is less than 1.5, and the purity is more than 95%, so that the polyhydroxyalkanoate obtained by the method through fractional purification has higher purity, uniform and accurate molecular weight, narrower molecular weight distribution and higher active terminal.
Drawings
FIG. 1 shows the molecular NMR hydrogen spectrum and NMR carbon spectrum of poly-3-hydroxybutyrate-co-4-hydroxybutyrate as the starting material.
FIG. 2 is a schematic view of a reaction reflux apparatus for the method of molecular weight fractionation and purification of polyhydroxyalkanoate of the present invention.
FIG. 3 is a comparison graph of permeation gel chromatography of poly 3-hydroxybutyrate-co-4 hydroxybutyrate starting material and molecular weight fractionated and purified poly 3-hydroxybutyrate.
Detailed Description
The endpoints of the ranges and any values disclosed herein are limited to the precise range or value. For ranges of values, one or more new ranges of values may be obtained by combining the endpoints of each range, the endpoints of each range and the individual values, and the individual values with each other, and these ranges of values are considered to be specifically disclosed herein.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The poly-3-hydroxybutyrate-co-4 hydroxybutyrate starting material of the following examples is a commercially available biofermentation resin, specifically, a commercially available bio-fermentation resin, such as the commercially available Maillard Producer science and technology Standard grade P34HB, having a molecular weight of greater than 20 million, a dispersity of greater than 1.8, and a molecular formula as shown in FIG. 1b, wherein the molecular structure is determined by the molecular NMR Hydrogen Spectrum of FIG. 1a and the molecular NMR carbon Spectrum of FIG. 1 b.
Molecular weight fractionation and purification were carried out using a reaction reflux apparatus as shown in FIG. 2 in the following examples, which included a two-necked flask 100 serving as a reaction vessel, a condenser 200 connected to one end of the two-necked flask 100 for refluxing a good solvent, and a dryer 300 provided at the other end of the condenser.
Example 1
The embodiment provides a method for grading and purifying the molecular weight of polyhydroxyalkanoate, wherein poly-3-hydroxybutyrate-co-4 hydroxybutyrate is used as a raw material to be graded and purified in the molecular weight, the ratio of a 3-hydroxybutyrate monomer to a 4-hydroxybutyrate monomer is (4-1): (1-4), the purity of the obtained poly-3-hydroxybutyrate-co-4 hydroxybutyrate is characterized by adopting liquid phase permeation gel chromatography (GPC), nuclear magnetic resonance hydrogen spectrum (H-NMR) and Thermogravimetry (TGA), the method for characterizing the initial poly-3-hydroxybutyrate-co-4 hydroxybutyrate in H-NMR is that 3mg of the poly-3-hydroxybutyrate-co-4 hydroxybutyrate raw material is dissolved in 0.6-1 mL of deuterated chloroform, under the strong scanning for 32-64 times, if the hydrogen spectrum does not contain impurity peaks, the poly 3-hydroxybutyrate-co-4-hydroxybutyrate with the purity of more than 85 percent is screened out.
The molecular weight grading and purifying method of the poly-3-hydroxybutyrate-co-4-hydroxybutyrate specifically comprises the following steps:
s1, mixing and dissolving 5g of the characterized poly 3-hydroxybutyrate-co-4-hydroxybutyrate raw material and 100ml of DMF for 6-24 h, dividing 1ml of water into 5 parts, adding one part of water into a reaction container every 5min, filling nitrogen gas to the upper part of the reaction container for protection, and heating at 120-140 ℃ for 12h for degradation;
s2, cooling the solution subjected to heating degradation in the step S1 to 30 ℃, mixing the solution with 2000ml of methanol, precipitating and separating out degradation products, and settling after 1-6 hours to completely collect the degradation products;
s3 degrading the degradation product collected in the step S2 with 500-10000 rad.s-1Centrifuging for 5min at the rotating speed, adding methanol for washing for 3-10 times in the centrifuging process, adding a proper amount of water, uniformly mixing, washing the degradation product by using ultrapure water for shaking for 5-10 min after centrifuging is finished, repeating for 5-10 times to ensure that all methanol is replaced, putting the washed degradation product into a rotary evaporator for water bath at 50-90 ℃, and carrying out rotary evaporation drying at the rotating speed of 20-100 rad.s < -1 > to obtain white powder, namely the poly-3-hydroxybutyrate-co-4-hydroxybutyrate subjected to molecular weight classification and purification.
Example 2
The embodiment provides a method for grading and purifying the molecular weight of polyhydroxyalkanoate, wherein poly-3-hydroxybutyrate-co-4 hydroxybutyrate is used as a raw material to be graded and purified in the molecular weight, the ratio of a 3-hydroxybutyrate monomer to a 4-hydroxybutyrate monomer is (4-1): (1-4), the purity of the obtained poly-3-hydroxybutyrate-co-4 hydroxybutyrate is characterized by adopting liquid phase permeation gel chromatography (GPC), nuclear magnetic resonance hydrogen spectrum (H-NMR) and Thermogravimetry (TGA), the method for characterizing the initial poly-3-hydroxybutyrate-co-4 hydroxybutyrate in H-NMR is that 3mg of the poly-3-hydroxybutyrate-co-4 hydroxybutyrate raw material is dissolved in 0.6-1 mL of deuterated chloroform, under the strong scanning for 32-64 times, if the hydrogen spectrum does not contain impurity peaks, the poly 3-hydroxybutyrate-co-4-hydroxybutyrate with the purity of more than 85 percent is screened out.
The molecular weight grading and purifying method of the poly-3-hydroxybutyrate-co-4-hydroxybutyrate specifically comprises the following steps:
s1 dissolving 5g of characterized poly 3-hydroxybutyrate-co-4-hydroxybutyrate raw material in 100ml of chloroform, and then dissolving 1ml of 12 mol. L-1The hydrochloric acid is divided into 3 parts, one part of hydrochloric acid is added into a reaction container every 5min, nitrogen is filled above the reaction container for protection, and the mixture is heated and refluxed for 12 hours at the temperature of 61 ℃ for degradation;
s2, cooling the solution after the heating degradation in the step S1 to 30 ℃, mixing the solution with 2000ml of methanol, precipitating and separating out the degradation product, and collecting the degradation product after the precipitation;
s3 degrading the product collected in S2 at 500-10000 rad.s-1Centrifuging for 2min at the rotating speed, adding methanol for washing for 3-10 times in the centrifuging process, adding a proper amount of water, uniformly mixing, washing the degradation product by using ultrapure water for 5-10 min after centrifuging, repeating for 5-10 times to ensure that all methanol is replaced, freezing the washed degradation product by using liquid nitrogen, putting the frozen degradation product into a freeze dryer, and freeze-drying for 12-72 h at the temperature of-20 ℃ and the vacuum degree of less than 100Pa to obtain the dried poly 3-hydroxybutyrate-co-4-hydroxybutyrate.
Example 3
This example analyzes the molecular weight of poly-3-hydroxybutyrate-co-4-hydroxybutyrate subjected to molecular weight fractionation and purification of example 1:
5mg of the molecular weight-fractionated and purified poly-3-hydroxybutyrate-co-4 hydroxybutyrate of example 1 and 5mg of the poly-3-hydroxybutyrate-co-4 hydroxybutyrate as starting materials were dissolved sufficiently after adding 1ml of DMF, respectively, and the resulting solution was subjected to liquid permeation gel chromatography (GPC) using a DMF mobile phase to measure the molecular weight of the polymer.
The analysis results are shown in FIG. 3, where the curve d-P34HB corresponds to the molecular weight of the polymer of poly-3-hydroxybutyrate-co-4 hydroxybutyrate fractionated and purified according to example 1, the curve P34HB corresponds to the molecular weight of the polymer of poly-3-hydroxybutyrate-co-4 hydroxybutyrate as the raw material, and as can be seen from FIG. 3, the abscissa value corresponding to the peak of the curve d-P34HB is smaller than the abscissa value corresponding to the peak of the curve P34HB, and the peak of the curve d-P34HB is narrower than the peak of the curve P34HB, which indicates that the poly-3-hydroxybutyrate-co-4 hydroxybutyrate with smaller molecular weight and narrower molecular weight distribution is successfully obtained after fractionation and purification according to the method of example 1.
Example 4
This example analyzes the purity of poly-3-hydroxybutyrate-co-4 hydroxybutyrate ester obtained by molecular weight fractionation and purification according to examples 1 and 2:
after 5g of the molecular weight-fractionated and purified poly-3-hydroxybutyrate-co-4 hydroxybutyrate of example 1 and 5g of the molecular weight-fractionated and purified poly-3-hydroxybutyrate-co-4 hydroxybutyrate of example 2 were dissolved in 100ml of chloroform and 1ml of water, respectively, they were heated under reflux at 61 ℃ for 72 hours to further degrade them, to obtain samples for purity measurement. The prepared samples of the embodiment 1 and the embodiment 2 are tested by a GC-2010Plus gas chromatograph, the initial temperature of the GC-2010Plus is set at 100 ℃, the retention time of the GC-2010Plus is set to be 1min at 100 ℃, then the temperature is increased to 280 ℃ according to the temperature rise rate of 30 ℃ per minute, the retention time of the GC-2010Plus is set to be 5min at 280 ℃, the whole detection process is 12min totally, after the detection is finished, the mass fraction of each monomer can be calculated according to the peak area of the product, then the molar ratio is calculated according to the mass fraction of each monomer, and the purity of the PHA sample to be detected can be calculated according to the amount of the added sample.
The above analysis shows that the purity of the poly-3-hydroxybutyrate-co-4 hydroxybutyrate raw material with the purity of only more than 85% in the embodiment 1 and the embodiment 2 is higher than 95% after primary molecular weight classification and purification by the method provided by the invention, so that the poly-3-hydroxybutyrate-co-4 hydroxybutyrate obtained by performing molecular weight classification and purification on the poly-3-hydroxybutyrate-co-4 hydroxybutyrate by the method provided by the invention has the advantages of smaller molecular weight and narrower molecular weight distribution, and the purity is also greatly improved.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.
Claims (10)
1. A method for molecular weight fractionation and purification of polyhydroxyalkanoate, comprising the steps of:
s1, dissolving the polyhydroxy fatty acid ester raw material in a good solvent, and adding a catalyst for heating degradation;
s2, cooling the solution after the degradation by heating in the step S1 to room temperature, mixing the solution with a poor solvent, precipitating and separating out the degradation product, and collecting the degradation product after the precipitation;
s3, after the degradation product collected in the step S2 is centrifuged, the degradation product is washed and dried, and thus the polyhydroxy fatty acid ester is subjected to molecular weight classification and purification.
2. The method of claim 1, wherein the polyhydroxyalkanoate is poly 3-hydroxybutyrate-co-4 hydroxybutyrate, the ratio of 3-hydroxybutyrate monomer to 4-hydroxybutyrate monomer of the poly 3-hydroxybutyrate-co-4 hydroxybutyrate is (1-10): (10-1), and the purity of the poly 3-hydroxybutyrate-co-4 hydroxybutyrate is 85% or more.
3. The method for fractionation and purification of polyhydroxyalkanoate according to claim 2, wherein the polyhydroxyalkanoate raw material has a number average and weight average relative molecular weight of 20 to 80 ten thousand, and a molecular weight dispersion degree of 1.5 to 2.5.
4. The method for molecular weight fractionation and purification of polyhydroxyalkanoate of claim 1, wherein in step S1, the good solvent is one or more of N, N-dimethylformamide, N-dimethylacetamide, chloroform, dichloromethane, or dioxane.
5. The method for molecular weight fractionation and purification of polyhydroxyalkanoate of claim 1, wherein in step S1, the catalyst is water, a weak base solution or a weak acid solution, and the mass ratio of the catalyst to the polyhydroxyalkanoate raw material is 1 (40-100).
6. The method for fractionation and purification of polyhydroxyalkanoate according to claim 1, wherein in step S1, the polyhydroxyalkanoate is dissolved in a good solvent for 2 to 48 hours, and is degraded by heating at 70 to 140 ℃ for 1 to 24 hours after adding a catalyst.
7. The method for molecular weight fractionation and purification of polyhydroxyalkanoate of claim 1, wherein in step S1, the catalyst is added to the solution of polyhydroxyalkanoate raw material and good solvent 1 to 10 times, and the time interval between each addition of the catalyst is 10 to 30 min.
8. The method for molecular weight fractionation and purification of polyhydroxyalkanoate of claim 1, wherein in step S2, the poor solvent is one or more mixtures of pure water, methanol or ethanol.
9. The method for molecular weight fractionation and purification of polyhydroxyalkanoate according to claim 1, wherein the mass ratio of the polyhydroxyalkanoate raw material to the good solvent is 1 (20 to 100).
10. The method for molecular weight fractionation and purification of polyhydroxyalkanoate according to claim 1, wherein the volume ratio of the good solvent to the poor solvent is 1 (30 to 200), and/or the mass ratio of the good solvent to the poor solvent is 1 (10 to 200).
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| CN114409886A (en) * | 2022-01-26 | 2022-04-29 | 珠海麦得发生物科技股份有限公司 | Purification method of polyhydroxy fatty acid ester |
| CN114428146A (en) * | 2022-01-27 | 2022-05-03 | 中国科学院宁波材料技术与工程研究所 | Method for analyzing molecular weight and distribution of polyhydroxyalkanoate by using gel permeation chromatography |
| CN114425101A (en) * | 2022-01-21 | 2022-05-03 | 北京大学口腔医学院 | Micro-nano double-layer structure antibacterial bracket and preparation method and application thereof |
| CN114591499A (en) * | 2022-03-17 | 2022-06-07 | 珠海麦得发生物科技股份有限公司 | Preparation method and application of poly (R) -3-hydroxybutyrate |
| CN114768564A (en) * | 2022-04-07 | 2022-07-22 | 珠海麦得发生物科技股份有限公司 | PHA purification device and purification method |
| CN114891196A (en) * | 2022-05-12 | 2022-08-12 | 珠海麦得发生物科技股份有限公司 | Injectable gel polyhydroxyalkanoate and preparation method and application thereof |
| CN115976088A (en) * | 2022-07-21 | 2023-04-18 | 深圳蓝晶生物科技有限公司 | Low endotoxin-containing fungus Roche and application thereof |
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| CN114425101A (en) * | 2022-01-21 | 2022-05-03 | 北京大学口腔医学院 | Micro-nano double-layer structure antibacterial bracket and preparation method and application thereof |
| CN114425101B (en) * | 2022-01-21 | 2022-07-22 | 北京大学口腔医学院 | Micro-nano double-layer structure antibacterial bracket and preparation method and application thereof |
| CN114409886A (en) * | 2022-01-26 | 2022-04-29 | 珠海麦得发生物科技股份有限公司 | Purification method of polyhydroxy fatty acid ester |
| CN114428146A (en) * | 2022-01-27 | 2022-05-03 | 中国科学院宁波材料技术与工程研究所 | Method for analyzing molecular weight and distribution of polyhydroxyalkanoate by using gel permeation chromatography |
| CN114591499A (en) * | 2022-03-17 | 2022-06-07 | 珠海麦得发生物科技股份有限公司 | Preparation method and application of poly (R) -3-hydroxybutyrate |
| CN114591499B (en) * | 2022-03-17 | 2023-06-13 | 珠海麦得发生物科技股份有限公司 | Preparation method and application of poly (R) -3-hydroxybutyrate |
| CN114768564A (en) * | 2022-04-07 | 2022-07-22 | 珠海麦得发生物科技股份有限公司 | PHA purification device and purification method |
| CN114891196A (en) * | 2022-05-12 | 2022-08-12 | 珠海麦得发生物科技股份有限公司 | Injectable gel polyhydroxyalkanoate and preparation method and application thereof |
| CN115976088A (en) * | 2022-07-21 | 2023-04-18 | 深圳蓝晶生物科技有限公司 | Low endotoxin-containing fungus Roche and application thereof |
| CN115976088B (en) * | 2022-07-21 | 2023-09-19 | 深圳蓝晶生物科技有限公司 | Low endotoxin content eutrophic rogowski bacteria and application thereof |
| CN116217964A (en) * | 2022-11-25 | 2023-06-06 | 北京科技大学 | Method for preparing low-dispersion index high-molecular weight polyvinyl chloride by centrifugal separation technology |
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