CN112094304A - Separation and purification method of enzymatic hydrolysis lignin - Google Patents
Separation and purification method of enzymatic hydrolysis lignin Download PDFInfo
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- CN112094304A CN112094304A CN202010991697.9A CN202010991697A CN112094304A CN 112094304 A CN112094304 A CN 112094304A CN 202010991697 A CN202010991697 A CN 202010991697A CN 112094304 A CN112094304 A CN 112094304A
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Abstract
The invention discloses a separation and purification method of enzymatic hydrolysis lignin, which comprises the following operation steps: s1: a separation measure; s2: chromatography of component A; s3: chromatography of component B; s4: analysis of component C. According to the method for separating and purifying the enzymatic hydrolysis lignin, the enzymatic hydrolysis lignin with a complex structure can be separated and purified, the reaction activity is improved, the utilization rate of the enzymatic hydrolysis lignin is improved, and petrochemical raw material synthetic materials can be replaced by the enzymatic hydrolysis lignin through separation and purification, so that energy is saved, and the energy crisis caused by petroleum shortage is solved.
Description
Technical Field
The invention belongs to the field of enzymolysis lignin research, and particularly relates to a separation and purification method of enzymolysis lignin.
Background
With the improvement of science and technology in China, the demand of China on petrochemical raw materials is more and more, the energy in China is also tense, the enzymolysis lignin is used as a byproduct for preparing fuel ethanol or functional sugar from plant straws, the yield is more and more large in recent years, but the reaction activity is lower due to the complexity of the structure of the enzymolysis lignin, and the utilization rate of the enzymolysis lignin is greatly limited. The literature shows that the substitution rate of the enzymatic hydrolysis lignin for replacing petrochemical raw material synthetic materials does not exceed 60 percent at present. Therefore, how to separate and purify the enzymatic hydrolysis lignin and improve the reaction activity becomes the key point of research.
However, the above solution has the following disadvantages in use: due to the complexity of the structure of the enzymatic hydrolysis lignin, the purification of the enzymatic hydrolysis lignin by a common purification method causes incomplete internal purification, and internal materials of the enzymatic hydrolysis lignin are wasted.
Disclosure of Invention
The invention aims to provide a separation and purification method of enzymatic hydrolysis lignin, which aims to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a separation and purification method of enzymatic hydrolysis lignin specifically comprises the following operation steps:
s1: separating, dissolving 50g of lignin in tetrahydrofuran with the mass 5 times of that of the lignin, sealing the mixture in a 500mL ground bottle, standing for 2-3h, dissolving for 3-4h under the assistance of ultrasonic waves, then carrying out vacuum filtration on the mixed solution, concentrating the solution part by using a rotary evaporator to obtain an extract, carrying out silica gel column chromatography on the extract, and eluting by using chloroform-acetone eluent to obtain a component A, B, C;
s2: performing chromatography on the component A, performing repeated silica gel column chromatography on the component A to obtain a compound A which mainly comprises a phenol derivative through gas chromatography analysis;
s3: performing chromatography on the component B, wherein the component B is subjected to repeated silica gel column chromatography and reversed-phase chromatographic column chromatography, and the obtained compound is a compound with a p-hydroxyphenyl structure generated by beta-5 or beta-1 bond breakage in a lignin molecular structure through nuclear magnetic resonance spectrum analysis;
s4: and (3) analyzing the component C, namely analyzing the component C into lipid or acid containing a benzene ring structure through gel Sephadex LH-20 and gas chromatography and liquid chromatography.
Preferably, the silica gel column chromatography comprises the following steps:
a. determining the ratio of chromatographic solutions, spotting the diluted sample on a silica gel plate by using a capillary, after a solvent is evaporated, respectively developing the sample by using the chromatographic solutions with different ratios, observing the position of the developed sample on the silica gel plate, and selecting the chromatographic solution used when the developed sample spot is positioned at 30-60% of the position of the plate;
b. dry-method sample loading, namely completely dissolving a certain mass of sample in a solvent, mixing 3 times of mass of silica gel with the sample, uniformly stirring, and removing the solvent by rotary evaporation to obtain a dry sample;
c. leaching the chromatographic solution, adding silica gel with a certain height into the glass column, compacting properly, pouring the dry sample into the uppermost layer, and leaching with the selected chromatographic solution at normal pressure;
d. collecting samples, filling the samples into 5ml sample bottles with 2ml samples after the chromatographic solution flows down with the samples, analyzing and combining the samples with the same components by passing the collected samples through a silica gel plate, and performing rotary evaporation on the chromatographic solution to obtain dry samples.
Preferably, the chloroform-acetone eluent in step S1 has a chloroform to acetone volume ratio of 30: 1.
Preferably, in step S2, component a is eluted sequentially through the following mixtures:
the volume ratio of the petroleum ether to the ethyl acetate is 4: 1;
the volume ratio of the petroleum ether to the acetone is 5: 1;
the volume ratio of chloroform to acetone is 30: 1.
Preferably, when the component B is subjected to silica gel column chromatography in the step S3, the elution is sequentially carried out through the following mixtures:
the volume ratio of the petroleum ether to the acetone is 8: 1;
the volume ratio of the petroleum ether to the ethyl acetate is 5: 1;
and in the step S3, the mass percent of the methanol and water system is 35-100% when the component B is subjected to reverse phase chromatography.
Preferably, the component C gel in step S4 is eluted through a mixture of: the volume ratio of chloroform to methanol is 1: 1.
The invention has the technical effects and advantages that:
according to the method for separating and purifying the enzymatic hydrolysis lignin, the enzymatic hydrolysis lignin with a complex structure can be separated and purified, the reaction activity is improved, the utilization rate of the enzymatic hydrolysis lignin is improved, and petrochemical raw material synthetic materials can be replaced by the enzymatic hydrolysis lignin through separation and purification, so that energy is saved, and the energy crisis caused by petroleum shortage is solved.
Drawings
FIG. 1 is a nuclear magnetic resonance spectrum of component B of a separation and purification method of enzymatic hydrolysis lignin;
FIG. 2 is a gas chromatogram of component C of a separation and purification method of enzymatic hydrolysis lignin.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A separation and purification method of enzymatic hydrolysis lignin specifically comprises the following operation steps:
s1: separating, dissolving 50g of lignin in tetrahydrofuran with the mass 5 times of that of the lignin, sealing the mixture in a 500mL ground bottle, standing the mixture for 2 hours, dissolving the mixture for 3 hours under the assistance of ultrasonic waves, then carrying out vacuum filtration on the mixed solution, concentrating the solution part by using a rotary evaporator to obtain an extract, carrying out silica gel column chromatography on the extract, and eluting the extract by using chloroform-acetone eluent to obtain a component A, B, C;
s2: performing chromatography on the component A, performing repeated silica gel column chromatography on the component A to obtain a compound A which mainly comprises a phenol derivative through gas chromatography analysis;
s3: performing chromatography on the component B, wherein the component B is subjected to repeated silica gel column chromatography and reversed-phase chromatographic column chromatography, and the obtained compound is a compound with a p-hydroxyphenyl structure generated by beta-5 or beta-1 bond breakage in a lignin molecular structure through nuclear magnetic resonance spectrum analysis;
s4: and (3) analyzing the component C, namely analyzing the component C into lipid or acid containing a benzene ring structure through gel Sephadex LH-20 and gas chromatography and liquid chromatography.
The silica gel column chromatography comprises the following steps:
a. determining the ratio of chromatographic solutions, spotting the diluted sample on a silica gel plate by using a capillary, after a solvent is evaporated, respectively developing the sample by using the chromatographic solutions with different ratios, observing the position of the developed sample on the silica gel plate, and selecting the chromatographic solution used when the developed sample spot is positioned at 30-60% of the position of the plate;
b. dry-method sample loading, namely completely dissolving a certain mass of sample in a solvent, mixing 3 times of mass of silica gel with the sample, uniformly stirring, and removing the solvent by rotary evaporation to obtain a dry sample;
c. leaching the chromatographic solution, adding silica gel with a certain height into the glass column, compacting properly, pouring the dry sample into the uppermost layer, and leaching with the selected chromatographic solution at normal pressure;
d. collecting samples, filling the samples into 5ml sample bottles with 2ml samples after the chromatographic solution flows down with the samples, analyzing and combining the samples with the same components by passing the collected samples through a silica gel plate, and performing rotary evaporation on the chromatographic solution to obtain dry samples.
The volume ratio of chloroform to acetone in the chloroform-acetone eluate in step S1 was 30: 1.
In step S2, component a is eluted through the following mixtures in sequence:
the volume ratio of the petroleum ether to the ethyl acetate is 4: 1;
the volume ratio of the petroleum ether to the acetone is 5: 1;
the volume ratio of chloroform to acetone is 30: 1.
When the component B in the step S3 is subjected to silica gel column chromatography, the following mixtures are sequentially used for elution:
the volume ratio of the petroleum ether to the acetone is 8: 1;
the volume ratio of the petroleum ether to the ethyl acetate is 5: 1;
and in the step S3, the mass percent of the methanol and water system is 35-100% when the component B is subjected to reverse phase chromatography.
The component C gel in step S4 was eluted through the following mixture: the volume ratio of chloroform to methanol is 1: 1.
The yield of the enzymatic hydrolysis lignin obtained by separating and purifying the enzymatic hydrolysis lignin is 80-90% (wherein the content ratio of the components A/B/C is 1:2:1), and according to different properties of the components of the enzymatic hydrolysis lignin, the components are applied to different types of reactions, so that the utilization rate of the enzymatic hydrolysis lignin reaches over 80%.
The method comprises the steps of dissolving the active ingredients in the substance by ultrasonic wave assistance, rapidly entering a solvent under the action of ultrasonic waves according to the existence state, polarity, solubility and the like of the active ingredients in the substance, thus obtaining a multi-component mixed extracting solution, separating, refining and purifying the extracting solution by a proper method, finally obtaining the required monomer chemical ingredients, separating according to different adsorption forces of the substance on silica gel by the separation principle of silica gel column chromatography, and analyzing the condition that the polarity of a mobile phase is in the polarity of a stationary phase by reversed-phase chromatography column chromatography.
According to the method for separating and purifying the enzymatic hydrolysis lignin, the enzymatic hydrolysis lignin with a complex structure can be separated and purified, the reaction activity is improved, the utilization rate of the enzymatic hydrolysis lignin is improved, and petrochemical raw material synthetic materials can be replaced by the enzymatic hydrolysis lignin through separation and purification, so that energy is saved, and the energy crisis caused by petroleum shortage is solved.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.
Claims (6)
1. A separation and purification method of enzymatic hydrolysis lignin is characterized in that: the method specifically comprises the following operation steps:
s1: separating, dissolving 50g of lignin in tetrahydrofuran with the mass 5 times of that of the lignin, sealing the mixture in a 500mL ground bottle, standing for 2-3h, dissolving for 3-4h under the assistance of ultrasonic waves, then carrying out vacuum filtration on the mixed solution, concentrating the solution part by using a rotary evaporator to obtain an extract, carrying out silica gel column chromatography on the extract, and eluting by using chloroform-acetone eluent to obtain a component A, B, C;
s2: performing chromatography on the component A, performing repeated silica gel column chromatography on the component A to obtain a compound A which mainly comprises a phenol derivative through gas chromatography analysis;
s3: performing chromatography on the component B, wherein the component B is subjected to repeated silica gel column chromatography and reversed-phase chromatographic column chromatography, and the obtained compound is a compound with a p-hydroxyphenyl structure generated by beta-5 or beta-1 bond breakage in a lignin molecular structure through nuclear magnetic resonance spectrum analysis;
s4: and (3) analyzing the component C, namely analyzing the component C into lipid or acid containing a benzene ring structure through gel Sephadex LH-20 and gas chromatography and liquid chromatography.
2. The method for separating and purifying enzymatic hydrolysis lignin according to claim 1, characterized in that: the silica gel column chromatography comprises the following steps:
a. determining the ratio of chromatographic solutions, spotting the diluted sample on a silica gel plate by using a capillary, after a solvent is evaporated, respectively developing the sample by using the chromatographic solutions with different ratios, observing the position of the developed sample on the silica gel plate, and selecting the chromatographic solution used when the developed sample spot is positioned at 30-60% of the position of the plate;
b. dry-method sample loading, namely completely dissolving a certain mass of sample in a solvent, mixing 3 times of mass of silica gel with the sample, uniformly stirring, and removing the solvent by rotary evaporation to obtain a dry sample;
c. leaching the chromatographic solution, adding silica gel with a certain height into the glass column, compacting properly, pouring the dry sample into the uppermost layer, and leaching with the selected chromatographic solution at normal pressure;
d. collecting samples, filling the samples into 5ml sample bottles with 2ml samples after the chromatographic solution flows down with the samples, analyzing and combining the samples with the same components by passing the collected samples through a silica gel plate, and performing rotary evaporation on the chromatographic solution to obtain dry samples.
3. The method for separating and purifying enzymatic hydrolysis lignin according to claim 1, characterized in that: the volume ratio of chloroform to acetone in the chloroform-acetone eluate in step S1 was 30: 1.
4. The method for separating and purifying enzymatic hydrolysis lignin according to claim 1, characterized in that: in step S2, component a is eluted through the following mixtures in sequence:
the volume ratio of the petroleum ether to the ethyl acetate is 4: 1;
the volume ratio of the petroleum ether to the acetone is 5: 1;
the volume ratio of chloroform to acetone is 30: 1.
5. The method for separating and purifying enzymatic hydrolysis lignin according to claim 1, characterized in that: when the component B in the step S3 is subjected to silica gel column chromatography, the following mixtures are sequentially used for elution:
the volume ratio of the petroleum ether to the acetone is 8: 1;
the volume ratio of the petroleum ether to the ethyl acetate is 5: 1;
and in the step S3, the mass percent of the methanol and water system is 35-100% when the component B is subjected to reverse phase chromatography.
6. The method for separating and purifying enzymatic hydrolysis lignin according to claim 1, characterized in that: the component C gel in step S4 was eluted through the following mixture: the volume ratio of chloroform to methanol is 1: 1.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1982322A (en) * | 2005-09-05 | 2007-06-20 | 福州大学 | Method for organically separating and extracting enzymic lignin |
| CN102277591A (en) * | 2011-08-02 | 2011-12-14 | 北京化工大学 | Method for electrochemically degrading lignin |
| CN102372607A (en) * | 2010-08-11 | 2012-03-14 | 中国科学院大连化学物理研究所 | Method for preparing single benzene ring phenolic compound from alkali lignin |
| CN102476980A (en) * | 2010-11-30 | 2012-05-30 | 中国科学院大连化学物理研究所 | Application of tungsten-based catalyst in lignin catalytic hydrogenation for producing aromatic compound |
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- 2020-09-19 CN CN202010991697.9A patent/CN112094304A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1982322A (en) * | 2005-09-05 | 2007-06-20 | 福州大学 | Method for organically separating and extracting enzymic lignin |
| CN102372607A (en) * | 2010-08-11 | 2012-03-14 | 中国科学院大连化学物理研究所 | Method for preparing single benzene ring phenolic compound from alkali lignin |
| CN102476980A (en) * | 2010-11-30 | 2012-05-30 | 中国科学院大连化学物理研究所 | Application of tungsten-based catalyst in lignin catalytic hydrogenation for producing aromatic compound |
| CN102277591A (en) * | 2011-08-02 | 2011-12-14 | 北京化工大学 | Method for electrochemically degrading lignin |
Non-Patent Citations (2)
| Title |
|---|
| 《国家执业药师手册》编辑委员会: "《国家执业药师手册》", 30 September 2002, 中国人事出版社 * |
| 刘建国，等: "《可再生能源导论》", 28 February 2017, 中国轻工业出版社 * |
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