CN107857762B - Method for extracting and preparing 6-benzylaminopurine in concentrated biogas slurry - Google Patents
Method for extracting and preparing 6-benzylaminopurine in concentrated biogas slurry Download PDFInfo
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- CN107857762B CN107857762B CN201711056708.9A CN201711056708A CN107857762B CN 107857762 B CN107857762 B CN 107857762B CN 201711056708 A CN201711056708 A CN 201711056708A CN 107857762 B CN107857762 B CN 107857762B
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D473/00—Heterocyclic compounds containing purine ring systems
- C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
- C07D473/32—Nitrogen atom
- C07D473/34—Nitrogen atom attached in position 6, e.g. adenine
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Abstract
The invention relates to the field of separation and preparation of components of concentrated biogas slurry, in particular to a method for extracting and preparing 6-benzylaminopurine from concentrated biogas slurry. The method specifically comprises the following steps: extracting the dried solid substance of the concentrated biogas liquid with methanol to obtain extract; dispersing the extract in distilled water to obtain suspension, and extracting with petroleum ether to obtain filter residue; mixing the ethyl acetate extract liquor and concentrating under reduced pressure to obtain dry solid; loading the dried solid on a silica gel column by a dry method, and using petroleum ether-ethyl acetate as an eluent according to the volume ratio of 20: 1; 10: 1; and 5:1, performing gradient elution, collecting fractions, detecting and merging fractions with the same spots, and separating and purifying to obtain the 6-benzylaminopurine. The purification method of the invention has simple operation, thereby having low operation cost, and having considerable economic and practical value and wide application prospect.
Description
Technical Field
The invention relates to the field of separation and preparation of components of concentrated biogas slurry, in particular to a method for extracting and preparing 6-benzylaminopurine from concentrated biogas slurry.
Background
Biogas slurry is a byproduct of biogas production fermentation, is called biological fertilizer and biological pesticide in agricultural production, and is widely popularized and applied. However, with the rapid development of biogas engineering in China, biogas slurry is also produced in large quantities, so the research on the recycling and harmless utilization technology of biogas slurry has important significance and is an effective way for realizing high-value utilization of biogas slurry. 6-benzylaminopurine is used as a broad-spectrum plant growth regulator produced by the plant in vivo, and plays an important role in promoting the biogas slurry as an organic fertilizer to crops. However, in the prior art, the existence of certain components in the biogas slurry is mainly measured by a high-calibration liquid phase when the biogas slurry is detected. The specific amounts of ingredients are not known. The method disclosed by the invention is used for separating the component from the biogas slurry, so that the content of the 6-benzylaminopurine is specifically known, a new method is provided for separating the component from the natural product, and a database of extraction and separation methods in the natural product chemistry is supplemented.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of an effective component (6-benzylaminopurine) in a concentrated biogas slurry, which comprises the following steps:
(1) and (4) leaching the dried solid substance of the concentrated biogas liquid with methanol to obtain extract.
Preferably, the method for drying the concentrated biogas slurry in the step (1) is to place the concentrated biogas slurry in a rotary evaporator for spin-drying.
Preferably, in the method for extracting the solid matter after the concentrated solution of the biogas slurry is dried by using the methanol in the step (1), the extraction time is 1-2 h, and the mass/volume ratio of the feed liquid is 150-.
Specifically, a proper amount of concentrated biogas slurry is taken and placed in a rotary evaporator for spin-drying, 200g of dried concentrated biogas slurry is taken, 1L of methanol is added, reflux extraction is carried out for 2h, extraction is carried out for 3 times, supernatant is taken for centrifugation, the supernatant is combined, reduced pressure concentration is carried out until the mixture is dried, and the mixture is weighed, so as to obtain the extract of the methanol extract.
(2) Dispersing the extract obtained in the step (1) in distilled water to prepare a suspension, and extracting with petroleum ether to obtain filter residue; specifically, the extract is dispersed in 1L distilled water to obtain suspension. Extracting with appropriate amount of petroleum ether for 3 times, and collecting the residue after petroleum ether extraction.
(3) Extracting the filter residue in the step (2) with ethyl acetate for several times (or 3 times), mixing the ethyl acetate extract liquor for several times, and concentrating under reduced pressure to obtain a dry solid;
specifically, the extract is extracted 3 times with ethyl acetate, and the ethyl acetate extract is concentrated under reduced pressure.
(4) Loading the dry solid obtained in the step (3) on a silica gel column by a dry method, and using petroleum ether-ethyl acetate as an eluent according to the volume ratio of 20: 1; 10: 1; 5:1, gradient elution is carried out, equal volume is set as one fraction collection (one fraction collection is set for every 30mL of cocoa), and after fractions of the same spots are detected and merged, separation and purification are carried out to obtain the 6-benzylaminopurine. Chloroform-methanol, petroleum ether-ethyl acetate, petroleum ether-acetone and other eluent systems and gradient elution in various proportions are tried, but finally, the collected fractions are found to have poor plate effect, dispersed points and light color and are basically in an invisible state. When trying to use petroleum ether-ethyl acetate as an eluent, the volume ratio is 20: 1; 10: 1; when gradient elution is carried out at 5:1, the point plate effect is obvious in an unexpected finding.
And (3) dissolving the dried solid obtained in the step (3) with a small amount of ethyl acetate, mixing the dissolved solid with 100-200-mesh crude silica gel, and scattering the mixture into a filled silica gel column after the ethyl acetate is volatilized.
Wherein, the silica gel column in the step (4) consists of 300-400-mesh fine silica gel.
Wherein, the spots obtained in the step (4) are spots obtained after detection by adopting thin-layer chromatography.
Specifically, after the dry solid is dissolved by a small amount of ethyl acetate, the 100-one 200-mesh crude silica gel and the dissolved ethyl acetate extract are weighed and mixed, stirring is carried out continuously, so that volatilization of ethyl acetate is facilitated, and the dry solid is reserved after the ethyl acetate is volatilized. Weighing quantitative 300-mesh 400-mesh fine silica gel, loading the silica gel into a glass column, scattering the sample after sample mixing into the loaded silica gel column by adopting a dry method, and then sequentially using petroleum ether: ethyl acetate 20: 1; 10: 1; and 5:1 and the like, collecting fractions in each 30mL, detecting by adopting Thin Layer Chromatography (TLC), combining fractions with the same spots, recovering an organic solvent to obtain three components a, b and c, and determining a component which is 6-benzylaminopurine by using an HPLC (high performance liquid chromatography) technology to obtain the purity of 98%. Concretely, petroleum ether: the ethyl acetate (20:1-5:1) section is divided into three components of a, b and c after TLC detection. Removing organic solvent from the three components, dissolving the three components by using chromatographic grade methanol, and detecting and determining the 6-benzylaminopurine by using an HPLC (high performance liquid chromatography) technology.
And (3) adding the obtained three components into an equivalent 6-benzylaminopurine standard substance respectively, and performing on a high performance liquid chromatography according to the HPLC method in the step (3) to obtain a liquid chromatogram of the three components and the 6-benzylaminopurine. Under the same conditions, the peak emergence time of the 6-benzylaminopurine standard substance is 47.449min, only one peak with the largest area is formed after the 6-benzylaminopurine standard substance is added into one component, the peak emergence time is 44.883min, and the peak area is greatly increased relative to a 6-benzylaminopurine chromatogram map, so that the conclusion can be drawn that the component prepared by the method is 6-benzylaminopurine.
Wherein, the specific parameters of the HPLC technology are as follows:
the instrument comprises the following steps: an Agilent-1260 liquid chromatograph (including a quaternary gradient pump, an autosampler, an ultraviolet detector, and an Agilent workstation); an oven;
reagent: ethyl acetate; petroleum ether; 100-200 mesh fine silica gel; 300-400 mesh fine silica gel; 6-benzylaminopurine standard; methanol (analytically pure and chromatographically pure); acetonitrile (chromatographically pure); ultrapure water
HPLC chromatographic conditions: mobile phase A: ultrapure water mobile phase B: acetonitrile (pure chromatogram)
Ultraviolet detection wavelength: 267nm
Flow rate: 0.3mL/min
Column temperature: 35 deg.C
The mobile phase elution gradient is shown in Table 1
TABLE 1 Linear gradient elution procedure for water and acetonitrile of test samples
The purification method of the invention has simple operation, thereby ensuring low operation cost. In addition, the yield of the 6-benzylaminopurine in the purification method is high, so that the purification method is suitable for large-scale industrial production, and the purified 6-benzylaminopurine has high purity, so that the purification method has considerable economic and practical value and wide application prospect. The recovery rate of the 6-benzylaminopurine prepared by the invention is obtained through the collected amount of the 6-benzylaminopurine, so that the content of the 6-benzylaminopurine in the biogas slurry is obtained. 6-benzylaminopurine is found to be one of the most main components in the biogas slurry and accounts for 11% of the effective components in the biogas slurry, although 6-benzylaminopurine has a promoting effect on plant growth, if the 6-benzylaminopurine is excessive, the growth of plants can be inhibited. Therefore, the knowledge of the content of the 6-benzylaminopurine can help a fertilizer applicator to prepare a more appropriate proportion to fertilize the crops, thereby better promoting the growth of the crops and promoting the development of agriculture.
Drawings
FIG. 1 is a liquid chromatogram of 6-benzylaminopurine standard. DAD1A, Sig 250,
4Ref＝off(085-0201.D)
FIG. 2 is a liquid chromatogram of a composition identified as 6 benzylaminopurine prepared using the present invention. DAD1A, Sig 250,4Ref off (085-0201.D)
Detailed Description
The invention will now be further illustrated by reference to the following examples:
the concentrated biogas slurry in the embodiment is a concentrated biogas slurry with application number of 201410284656.0 and prepared by an engineering preparation method.
(1) And (3) treating a biogas concentrate sample: placing a proper amount of concentrated biogas slurry in a rotary evaporator for spin-drying, taking 200g of dried concentrated biogas slurry, adding 1L of methanol, extracting under reflux for 2h, extracting for 3 times, taking supernatant, centrifuging, combining supernatants, concentrating under reduced pressure to dryness, weighing to obtain extract of methanol extract, and dispersing the extract in 1L of distilled water to obtain suspension. Extracting with appropriate amount of petroleum ether for 3 times, extracting the residue with appropriate amount of ethyl acetate for 3 times, and concentrating the ethyl acetate extractive solution under reduced pressure to dry to obtain ethyl acetate extract.
(2) Preparation of 6-benzylaminopurine in concentrated biogas slurry
Dissolving the ethyl acetate phase extract with a small amount of ethyl acetate, weighing 100-mesh 200-mesh crude silica gel and the dissolved ethyl acetate extract, stirring continuously to facilitate the volatilization of ethyl acetate, and standing after the ethyl acetate is volatilized. Weighing quantitative 300-mesh 400-mesh fine silica gel, loading the silica gel into a glass column, scattering the sample after sample mixing into the loaded silica gel column by adopting a dry method, and then sequentially using petroleum ether: ethyl acetate 20: 1; 10: 1; 5:1, etc., and collecting fractions every 30mL, detecting by Thin Layer Chromatography (TLC), combining fractions with the same spot, and recovering the organic solvent. Mixing petroleum ether: ethyl acetate (20:1-5:1) is separated into four components of a, b and c after TLC detection. Removing organic solvent from the three components, dissolving with chromatographic grade methanol, and detecting with HPLC technique.
(3) HPLC method for detecting separated product of biogas concentrate
The instrument comprises the following steps: an Agilent-1260 liquid chromatograph (including a quaternary gradient pump, an autosampler, an ultraviolet detector, and an Agilent workstation); an oven;
reagent: ethyl acetate; petroleum ether; 100-200 mesh fine silica gel; 300-400 mesh fine silica gel; 6-benzylaminopurine standard; methanol (analytically pure and chromatographically pure); acetonitrile (chromatographically pure); ultrapure water
HPLC chromatographic conditions: mobile phase A: ultrapure water mobile phase B: acetonitrile (pure chromatogram)
Ultraviolet detection wavelength: 267nm
Flow rate: 0.3mL/min
Column temperature: 35 deg.C
The mobile phase elution gradient is shown in Table 1
TABLE 1 Linear gradient elution procedure for water and acetonitrile of test samples
(4) And (3) respectively adding the three components obtained in the step (2) into an equal amount of 6-benzylaminopurine standard substance, and testing on a high performance liquid chromatography according to the HPLC method in the step (3) to obtain liquid chromatogram maps of the three components and 6-benzylaminopurine. Under the same conditions, the peak emergence time of the 6-benzylaminopurine standard substance is 47.449min, only one peak with the largest area is formed after the 6-benzylaminopurine standard substance is added into one component, the peak emergence time is 44.883min, and the peak area is greatly increased relative to a 6-benzylaminopurine chromatogram map, so that the conclusion can be drawn that the component prepared by the method is 6-benzylaminopurine.
The purity of 6-benzylaminopurine was determined to be 98%.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention, and the present invention should not be limited by the disclosure of the preferred embodiments. Therefore, it is intended that all equivalents and modifications which do not depart from the spirit of the invention disclosed herein are deemed to be within the scope of the invention.
Claims (8)
1. A method for extracting and preparing 6-benzylaminopurine in concentrated biogas slurry is characterized by comprising the following steps:
(1) extracting the dried solid substance of the concentrated biogas liquid with methanol to obtain extract;
(2) dispersing the extract obtained in the step (1) in distilled water to prepare a suspension, and extracting with petroleum ether to obtain filter residue;
(3) extracting the filter residue in the step (2) with ethyl acetate for a plurality of times, mixing the ethyl acetate extract liquor for a plurality of times, and concentrating under reduced pressure to obtain a dry solid;
(4) loading the dry solid obtained in the step (3) on a silica gel column by a dry method, and using petroleum ether-ethyl acetate as an eluent according to the volume ratio of 20: 1; 10: 1; and (5) performing gradient elution at the ratio of 1: 1, setting equal volume as one fraction for collection, detecting and merging fractions with the same spots, and separating and purifying to obtain the 6-benzylaminopurine.
2. The extraction preparation method according to claim 1, wherein: the method for drying the biogas slurry concentrated solution in the step (1) is to place the biogas slurry in a rotary evaporator for spin-drying.
3. The extraction preparation method according to claim 1, wherein: in the method for extracting the solid matters from the dried biogas slurry by using the methanol in the step (1), the extraction time is 1-2 h, and the mass/volume ratio of the material liquid is 150-250 g/L.
4. The extraction preparation method according to claim 1, wherein: and (3) extracting the filter residue in the step (2) with ethyl acetate for 3 times.
5. The extraction preparation method according to claim 1, wherein: and (4) dissolving the dried solid in the step (3) with a small amount of ethyl acetate, mixing the dissolved solid with 100-200-mesh crude silica gel, and scattering the mixture into a filled silica gel column after the ethyl acetate is volatilized.
6. The extraction preparation method according to claim 1, wherein: the silica gel column in the step (4) consists of 300-400-mesh fine silica gel.
7. The extraction preparation method according to claim 1, wherein: the set of 50ml for step (4) was one fraction collected.
8. The extraction preparation method according to claim 1, wherein: the spots obtained in the step (4) are spots obtained by adopting thin-layer chromatography detection.
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| CN201711056708.9A CN107857762B (en) | 2017-10-27 | 2017-10-27 | Method for extracting and preparing 6-benzylaminopurine in concentrated biogas slurry |
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| ES2279719B1 (en) * | 2006-01-26 | 2008-07-16 | Timac Agro España, S.A. | NEW METABOLIC AND NUTRITIONAL ACTIVATOR FOR PLANTS. |
| CN104058799B (en) * | 2014-06-24 | 2016-09-07 | 山东民和生物科技股份有限公司 | A kind of biogas slurry concentrated solution and through engineering approaches preparation method |
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