CN113292713B - Molecular weight grading and purifying method of polyhydroxyalkanoate - Google Patents
Molecular weight grading and purifying method of polyhydroxyalkanoate Download PDFInfo
- Publication number
- CN113292713B CN113292713B CN202110637249.3A CN202110637249A CN113292713B CN 113292713 B CN113292713 B CN 113292713B CN 202110637249 A CN202110637249 A CN 202110637249A CN 113292713 B CN113292713 B CN 113292713B
- Authority
- CN
- China
- Prior art keywords
- polyhydroxyalkanoate
- molecular weight
- hydroxybutyrate
- purification
- degradation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- 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
-
- 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
-
- 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
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/62—Plastics recycling; Rubber recycling
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Sustainable Development (AREA)
- Polyesters Or Polycarbonates (AREA)
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 a polyhydroxyalkanoate raw material in a good solvent, and adding a catalyst for heating degradation; s2, cooling the solution subjected to the heating degradation in the step S1 to room temperature, mixing the solution with a poor solvent, precipitating and separating out a degradation product, and collecting the degradation product after the degradation product is precipitated; and S3, centrifuging the degradation product collected in the step S2, and washing and drying the degradation product to finish molecular weight classification and purification of the polyhydroxyalkanoate. 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 particularly excellent in performance, and a nerve conduit made of PHA, a cartilage material and an esophagus regeneration auxiliary functional material are reported, so that good biocompatibility and excellent physicochemical and mechanical properties are proved, poly-4-hydroxybutyrate (P4 HB) in the PHA family is used as a patch for repairing abdominal wall defects of a human body, the patch has better biocompatibility compared with a traditional polypropylene (PP) patch, but has the defect of high cost, and related products are not provided at home, the main reason is the difficulty in preparing medical grade polyester, namely the requirement on molecular weight is relatively accurate, the requirement on purity is high, poly-3-hydroxybutyrate-co-4-hydroxybutyrate (P34 HB) is the most excellent product in performance in the PHA family, and the degradation product of the poly-3-hydroxybutyrate is 3-hydroxybutyrate, so that the repair of biological tissues is facilitated. And the proportion of the 3-hydroxybutyric acid to 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 from PHA obtained by biological fermentation to PHA collected and purified and to PHA processing devices, for example, two kinds of salt-tolerant bacteria have been successfully obtained by strict screening by the Chen national strong group of Qinghua university in China, the large-scale production of a series of PHA is realized by microbial fermentation, medical-grade PHA is obtained by improving the purification method, if the traditional polyolefin plastic is replaced, the white pollution caused by chemical engineering plastic can be effectively solved, however, as the biological fermentation is insufficient relative to the chemical process, the basic chemical parameters such as the product molecular weight are difficult to control, the subsequent purification process often contains residues of cell membranes and cytoplasm, and also contains certain cellular endotoxin, the obtained PHA raw material is not pure enough, and the 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 view of the problem of low PHA purity, the chinese patent "a purification method of polyhydroxyalkanoate" (application No. 201910001260.3, published: 2019.03.26) discloses a purification method of polyhydroxyalkanoate comprising the steps of: (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, and 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 disadvantages of the prior art and providing a method for fractionating and purifying polyhydroxyalkanoate, which has simple steps and convenient operation, and can fractionate the polyhydroxyalkanoate while purifying it, and the polyhydroxyalkanoate obtained by fractionation and purification by this method has high purity, uniform and precise 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 a polyhydroxyalkanoate raw material in a good solvent, and adding a catalyst for heating degradation;
s2, cooling the solution subjected to the heating degradation in the step S1 to room temperature, mixing the solution with a poor solvent, precipitating and separating out a degradation product, and collecting the degradation product after the degradation product is precipitated;
and S3, centrifuging the degradation product collected in the step S2, and washing and drying the degradation product to finish molecular weight classification and purification of the polyhydroxyalkanoate.
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 molecular weight fractional regulation and purification of the polyhydroxyalkanoate raw material can be realized in a shorter time, the solution after the heating degradation is mixed with a poor solution, the polyhydroxyalkanoate is separated out by utilizing the lower solubility between the polyhydroxyalkanoate and the poor solvent, other materials such as cell debris, pigments or impure polyhydroxyalkanoate are dissolved in the poor solvent, the separated polyhydroxyalkanoate can be collected by utilizing centrifugation, and the collection speed is accelerated.
Preferably, in step S2, the solution after the thermal degradation in step S1 is cooled to room temperature of 30 ℃.
Preferably, the time for precipitation 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 polyhydroxyalkanoate with 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 to 100).
Further, in the 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 30min.
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 molecular chain stretching is facilitated, and higher yield can be obtained.
In the technical scheme, the reaction temperature should be 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 rotational speed of the centrifuge during the centrifugal separation is 500 to 10000rad · S -1 The centrifugation time is 2-30 min.
Preferably, in the centrifugation 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 to 6 hours, and the time for each subsequent precipitation is 0.5 to 5 hours.
Preferably, the degradation product is finally washed 5 to 10 times with pure water.
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 at-20 ℃ under a vacuum of less than 100Pa for 12-72 h to ensure sample dryness.
More preferably, the degradation products are subjected to water bath at 50-90 ℃ and rotary evaporation at a rotation speed of 20-100 rad s < -1 >.
Compared with the prior art, the invention has the beneficial effects that:
the method has 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 1-10 ten thousand, namely 1-20 g.mol -1 And 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 and narrower molecular weight distributionAnd has a higher active end.
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 in the following examples is a commercially available biofermentation resin, specifically, a commercially available standard grade P34HB having a molecular weight of greater than 20 ten thousand, a dispersity of greater than 1.8, and a molecular formula as shown in fig. 1b, and its 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
This example provides a method for classifying and purifying molecular weight of polyhydroxyalkanoate, where poly-3-hydroxybutyrate-co-4 hydroxybutyrate is used as a raw material, the ratio of 3-hydroxybutyrate monomer to 4-hydroxybutyrate monomer is (4-1): (1-4), and the purity of the obtained poly-3-hydroxybutyrate-co-4 hydroxybutyrate is characterized by liquid phase permeation gel chromatography (GPC), nuclear magnetic resonance hydrogen spectrum (H-NMR) and Thermogravimetry (TGA), and specifically, the method for characterizing the initial poly-3-hydroxybutyrate-co-4 hydroxybutyrate in H-NMR is to dissolve 3mg of poly-3-hydroxybutyrate-co-4 hydroxybutyrate raw material in 0.6-1 mL of deuterated chloroform, and select a poly-3-hydroxybutyrate-co-4 hydroxybutyrate with a purity of greater than 85% if the hydrogen spectrum does not contain an impurity peak under strong scanning for 32-64 times.
The molecular weight grading and purifying method of the poly-3-hydroxybutyrate-co-4-hydroxybutyrate specifically comprises the following steps:
s1, mixing 5g of a characterized poly 3-hydroxybutyrate-co-4 hydroxybutyrate raw material and 100ml of DMF (dimethyl formamide) for dissolving for 6-24 h, dividing 1ml of water into 5 parts, adding one part of water into a reaction vessel every 5min, filling nitrogen gas above the reaction vessel for protection, and heating at 120-140 ℃ for 12h for degradation;
s2, cooling the solution subjected to the heating degradation in the step S1 to 30 ℃, mixing the solution with 2000ml of methanol, precipitating and separating out a degradation product, and settling after 1-6 hours to completely collect the degradation product;
s3, degrading products collected in the step S2 by 500-10000 rad.s -1 Centrifuging 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 the methanol is completely replaced, putting the washed degradation product into a rotary evaporator for water bath at the temperature of 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
This example provides a method for classifying and purifying molecular weight of polyhydroxyalkanoate, wherein poly-3-hydroxybutyrate-co-4 hydroxybutyrate is used as a raw material, the ratio of 3-hydroxybutyrate monomer to 4-hydroxybutyrate monomer is (4-1): (1-4), and the purity of the obtained poly-3-hydroxybutyrate-co-4 hydroxybutyrate is characterized by using liquid phase permeation gel chromatography (GPC), nuclear magnetic resonance hydrogen spectroscopy (H-NMR) and Thermogravimetry (TGA), specifically, the characterization method of the initial poly-3-hydroxybutyrate-co-4 hydroxybutyrate in H-NMR is that 3mg of poly-3-hydroxybutyrate-co-4 hydroxybutyrate raw material is dissolved in 0.6-1 mL of deuterated chloroform, and under the strong scanning for 32-64 times, the hydrogen spectrum does not contain impurity peaks, which indicates that poly-3-hydroxybutyrate-co-4 hydroxybutyrate with purity higher than 85% 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 -1 The 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 subjected to the heating degradation in the step S1 to 30 ℃, mixing the solution with 2000ml of methanol, precipitating and separating out degradation products, and collecting the degradation products after the degradation products are precipitated;
s3, degrading products collected in the step S2 by the amount of 500-10000 rad.s -1 Centrifuging 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, shaking and washing the degradation product for 5-10 min by using ultrapure water after centrifuging is finished, 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 minus 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 poly-3-hydroxybutyrate-co-4 hydroxybutyrate and 5mg of the poly-3-hydroxybutyrate-co-4 hydroxybutyrate obtained by molecular weight fractionation and purification in example 1 were taken as raw materials, and after adding 1ml of DMF, they were sufficiently dissolved, and the resulting mixture was subjected to liquid phase permeation gel chromatography (GPC) using DMF as a mobile phase to measure the molecular weight of the polymer.
As shown in FIG. 3, the curve d-P34HB corresponds to the molecular weight of the polymer of poly-3-hydroxybutyrate-co-4 hydroxybutyrate obtained by molecular weight fractionation and purification in example 1, the curve P34HB corresponds to the molecular weight of the polymer as the raw material of poly-3-hydroxybutyrate-co-4 hydroxybutyrate, 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 poly-3-hydroxybutyrate-co-4 hydroxybutyrate with smaller molecular weight and narrower molecular weight distribution can be successfully obtained by fractionation and purification by 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 (9)
1. A method for classifying and purifying the molecular weight of polyhydroxyalkanoate is characterized by comprising the following steps:
s1, dissolving a polyhydroxyalkanoate raw material in a good solvent, adding a catalyst for heating degradation, wherein 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);
s2, cooling the solution subjected to the heating degradation in the step S1 to room temperature, mixing the solution with a poor solvent, precipitating and separating out a degradation product, and collecting the degradation product after the degradation product is precipitated;
and S3, centrifuging the degradation product collected in the step S2, and washing and drying the degradation product to finish molecular weight classification and purification of the polyhydroxyalkanoate.
2. The method for molecular weight fractionation and purification of polyhydroxyalkanoate according to claim 1, wherein the polyhydroxyalkanoate raw material 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 to 10): (10 to 1), and the purity of the poly 3-hydroxybutyrate-co-4 hydroxybutyrate is 85% or more.
3. The method for classifying and purifying the molecular weight 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 fractionation and purification of a polyhydroxyalkanoate by molecular weight 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 a polyhydroxyalkanoate according to claim 1, wherein in step S1, the polyhydroxyalkanoate is dissolved in a good solvent for 2 to 48h, and is degraded by heating at 70 to 140 ℃ for 1 to 24h after a catalyst is added.
6. The method for molecular weight fractionation and purification of polyhydroxyalkanoate according to claim 1, wherein in step S1, the catalyst is added to the solution of polyhydroxyalkanoate raw material and a good solvent 1 to 10 times, and the time interval between each addition of the catalyst is 10 to 30min.
7. The method for molecular weight fractionation and purification of polyhydroxyalkanoate according to claim 1, wherein in step S2, the poor solvent is one or more mixtures of pure water, methanol or ethanol.
8. The method for fractionation and purification of a polyhydroxyalkanoate according to claim 1, wherein the mass ratio of the polyhydroxyalkanoate raw material to the good solvent is 1 (20 to 100).
9. The method for fractionation and purification of a 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).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110637249.3A CN113292713B (en) | 2021-06-08 | 2021-06-08 | Molecular weight grading and purifying method of polyhydroxyalkanoate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110637249.3A CN113292713B (en) | 2021-06-08 | 2021-06-08 | Molecular weight grading and purifying method of polyhydroxyalkanoate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113292713A CN113292713A (en) | 2021-08-24 |
| CN113292713B true CN113292713B (en) | 2022-10-25 |
Family
ID=77327573
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110637249.3A Active CN113292713B (en) | 2021-06-08 | 2021-06-08 | Molecular weight grading and purifying method of polyhydroxyalkanoate |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113292713B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN114768564B (en) * | 2022-04-07 | 2024-05-28 | 珠海麦得发生物科技股份有限公司 | PHA (polyhydroxyalkanoate) purification device and purification method |
| CN114891196B (en) * | 2022-05-12 | 2023-07-14 | 珠海麦得发生物科技股份有限公司 | Injectable gel polyhydroxyalkanoate and preparation method 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 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7361725B2 (en) * | 2004-05-18 | 2008-04-22 | Ga-Er Yu | Process of producing low molecular weight poly(hydroxyalkanoate)s from high molecular weight poly(hydroxyalkanoate)s |
| JP2008189861A (en) * | 2007-02-07 | 2008-08-21 | Tosoh Corp | Poly(3-hydroxy butyrate)-based polymer excellent in color tone and method for purification thereof |
| WO2013049161A1 (en) * | 2011-09-27 | 2013-04-04 | Tepha, Inc. | Controlled hydrolysis of poly-4-hydroxybutyrate and copolymers |
| CN109517156A (en) * | 2019-01-02 | 2019-03-26 | 清华大学 | A kind of purification process of polyhydroxyalkanoate |
-
2021
- 2021-06-08 CN CN202110637249.3A patent/CN113292713B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN113292713A (en) | 2021-08-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113292713B (en) | Molecular weight grading and purifying method of polyhydroxyalkanoate | |
| US7893194B2 (en) | Single solvent polymer extraction methods | |
| CN107163204B (en) | A kind of preparation method and application of high-strength and high ductility thermosetting resin based composites | |
| CN109517156A (en) | A kind of purification process of polyhydroxyalkanoate | |
| CN114478468B (en) | Method for refining glycolide and glycolide obtained by same | |
| CN115536821A (en) | Preparation method of bisphenol B/phenol red type copolymerized aryl ester material film | |
| Noritake et al. | Recycling of polyethylene terephthalate using high-pressure steam treatment | |
| Magagnini et al. | Synthesis of PP–LCP graft copolymers and their compatibilizing activity for PP/LCP blends | |
| CN114409886A (en) | Purification method of polyhydroxy fatty acid ester | |
| CN109280158B (en) | Method for producing poly D-lactic acid by ring-opening polymerization of D-lactide | |
| CN114573801A (en) | Preparation method of phenol red type polyarylate material | |
| CN101544751B (en) | Biodegradable water-soluble polycarbonate and method for preparing same | |
| JPWO2005049692A1 (en) | Process for producing polyhydroxyalkanoate crystals | |
| CN112029122B (en) | Biodegradable film and preparation method thereof | |
| Ravi et al. | Synthesis and characterization of certain biodegradable random aliphatic copolyesters | |
| CN114591499B (en) | Preparation method and application of poly (R) -3-hydroxybutyrate | |
| Wang et al. | Synthesis and characterization of poly (L-glutamic acid-co-L-aspartic acid) | |
| CN109337052B (en) | Method for producing poly-L-lactic acid by ring-opening polymerization of L-lactide | |
| EP4153654B1 (en) | Process for isolating pha from a pha-rich bacterial biomass | |
| CN115746292B (en) | Extraction method of phycocyanin in activated sludge | |
| US7153928B2 (en) | Method for producing polyhydroxyalkanoate crystal | |
| CN115926136A (en) | Continuous production process of carbon dioxide-based biodegradable polymer | |
| Chen et al. | Study on Recrystallization Process of Poly (DL-lactide)/Poly (ethylene glycol) Diblock Copolymer | |
| CN118005903A (en) | Preparation method of polybutylene succinate with low extractables content | |
| CN114621179A (en) | Method for synthesizing glycolide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |