CN112284841A - Method for quickly removing ethanol in chloroform - Google Patents
Method for quickly removing ethanol in chloroform Download PDFInfo
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- CN112284841A CN112284841A CN201910674972.1A CN201910674972A CN112284841A CN 112284841 A CN112284841 A CN 112284841A CN 201910674972 A CN201910674972 A CN 201910674972A CN 112284841 A CN112284841 A CN 112284841A
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Abstract
The invention relates to the field of biochemistry, in particular to a method for quickly removing ethanol in chloroform. Standing and adsorbing chloroform to be treated by alumina particles; thus obtaining chloroform with ethanol removed. Compared with the currently generally adopted liquid separation distillation method, the method disclosed by the invention is simple and rapid to operate, does not need heating distillation, reduces volatilization and decomposition of chloroform in the experimental process, and greatly reduces pollution to a laboratory and toxicity to experimenters.
Description
Technical Field
The invention relates to the field of biochemistry, in particular to a method for quickly removing ethanol in chloroform.
Background
Soil microorganisms are important components of an ecological system and play a key role in maintaining biological diversity, organic matter decomposition, nutrient conversion and the like of the ecological system. The microbial biomass of carbon, nitrogen and phosphorus is an important index for researching soil microbes and the material circulation of an ecosystem.
The most common method for determining soil microbial biomass is the chloroform fumigation leaching method. The key point of the method is that the soil and the chloroform without ethanol are placed in a vacuum drier and pumped by an air pump until the chloroform is boiled, and then the vacuum state is kept. After the soil sample is fumigated by chloroform (1-3 days), the dead cells of the soil microorganisms are cracked, and microorganisms such as carbon, nitrogen and phosphorus are released. Then, chloroform is easily oxidized into chlorine, hydrogen chloride and virulent phosgene carbonyl chloride under sunlight, and 1 percent of ethanol is used as a stabilizer for the commercially supplied chloroform so as to eliminate the generated phosgene. Therefore, ethanol-free chloroform needs to be prepared by itself before the determination of the amount of soil microorganisms.
The existing method for preparing ethanol-free chloroform in a laboratory is a liquid separation distillation method. The most common methods are: 500ml of chloroform was added to a 1000ml separatory funnel, 50ml of a 5% sulfuric acid solution was added thereto, followed by shaking sufficiently, and the lower sulfuric acid solution was discarded, and the procedure was repeated 3 times. 50ml of deionized water was added thereto, shaken up, and the upper layer of water was discarded, followed by 5 times. Transferring the lower layer of chloroform into a distillation flask, and distilling in a water bath at 62 deg.C to obtain an effluent which is ethanol-free chloroform. The distilled ethanol-free chloroform was stored in a brown bottle, and 20g of potassium carbonate was added thereto and stored under refrigeration for use. It can be seen that the liquid-separating distillation method has many steps, wastes time and labor, and takes 6-9 hours to prepare ethanol-free chloroform once. In addition, in a long experiment process, particularly in a high-temperature distillation process, volatilization and decomposition of chloroform pollute a laboratory and generate strong toxic action on experimenters.
At present, no other method is found in China to remove ethanol in chloroform besides the liquid-separating distillation method. Therefore, finding a simple and rapid method for removing ethanol in chloroform is an urgent problem to be solved in the soil microbial load measuring process.
Disclosure of Invention
The invention provides a method for quickly and simply removing ethanol in chloroform; the method is suitable for preparing ethanol-free chloroform in the soil microorganism biomass carbon, nitrogen and phosphorus determination process.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for rapidly removing ethanol in chloroform comprises standing chloroform to be treated by alumina particles for adsorption; thus obtaining chloroform with ethanol removed.
The volume mass ratio of the chloroform to the alumina particles is 5-30: 100.
The alumina particles were analytical grade, 100-200 mesh.
Injecting the chloroform to be treated into a chromatographic column filled with alumina particles, and standing for 30-40min after injection; then the concentration is controlled to be 20-25 ml.min-1The flow rate of the chloroform was measured and the effluent was collected, i.e., the chloroform from which the ethanol was removed.
The chromatographic column is filled with an alumina particle layer, and the upper surface and the lower surface of the particle layer are covered with filter paper.
The filter paper was wetted with chloroform.
The ethanol-free chloroform prepared by the invention is mainly applied to fumigation for killing soil microorganisms in the process of measuring the carbon, nitrogen and phosphorus contents of the soil microorganisms.
Compared with the existing liquid separation distillation method, the method has the advantages that:
(1) the equipment and reagents required for the experiment are simple. The invention relates to the method for removing ethanol in chloroform by using activated alumina, which only needs one glass chromatographic column and activated alumina particles. Therefore, compared with the existing liquid separation distillation method, the required experimental equipment is greatly reduced, and the use of hazardous chemical sulfuric acid is reduced.
(2) The time required for the experiment was short. The time required by the ethanol-free chloroform obtained by the invention is 30-40min, while the prior liquid separation distillation method requires 6-9 h. Thus, the present invention greatly shortens the time required to prepare ethanol-free chloroform. Can be prepared within 1 hour before the microbial biomass is measured, and is used for purification.
(3) The toxic effect generated in the experimental process is greatly reduced. The chloroform is oxidized and decomposed by light to generate virulent phosgene carbonyl chloride, which has great toxicity to experimenters. Compared with the prior method, the time required for preparing the ethanol-free chloroform is shortened by more than ten times, so that the volatilization and decomposition amount of the chloroform in the chloroform purification process is greatly reduced, and the chloroform can be purified before use before experiment, thereby greatly reducing the pollution to a laboratory and the toxic action of experimenters.
Drawings
FIG. 1 is a schematic view of a glass chromatographic column according to an embodiment of the present invention.
Detailed description of the preferred embodiment
The invention is further illustrated by the following examples
Example 1
Ethanol-free chloroform:
(1) the activated alumina chromatographic column is commercially available, and as shown in figure 1, the glass chromatographic column has an outer diameter of 350mm, an effective length of 250mm, 4 triangular glass plates at the bottom, a glass piston and a glass plug at the inner grinding opening at the top.
The chromatographic column is placed on an iron stand, a glass piston is closed, 2 layers of filter paper which is slightly wetted by chloroform are paved at the bottom end of the column, then analytically pure active alumina particles are slowly filled into a chromatographic column with the size of 350mm multiplied by 250mm, and 2 layers of filter paper are paved at the upper end of the alumina particles.
(2) The amount of activated alumina to achieve the best removal effect was determined by setting a gradient experiment:
firstly, arranging and filling 6 gradient experimental active alumina chromatographic columns according to the mode; wherein, 6 gradients are respectively filled with 5, 10, 15, 20, 25 and 30g of alumina.
② accurately measuring 6 parts of chloroform containing 100ml of ethanol in a brown bottle, then slowly pouring each part of chloroform into 6 chromatographic columns along a glass rod, covering plugs of the chromatographic columns, standing for 30min, then taking down the plugs at the upper ends of the chromatographic columns, opening a piston at the lower end to enable the chloroform to flow for 20 ml.min-1The flow rate was passed through the column and collected in the lower brown reagent bottle.
③ separately taking 25ml of chloroform collected under each chromatographic column, placing the chloroform in 50ml beakers, and placing a small spoon of artificial zeolite in each beaker. And (4) putting the beaker into a dryer, sealing the dryer, exhausting air by using a vacuum pump, and observing the boiling change of the chloroform.
And fourthly, after vacuumizing for 50s, simultaneously boiling 6 chromatographic columns to meet the requirement of keeping the boiling time for 3min in microbial chloroform fumigation, and further removing ethanol in chloroform containing ethanol adsorbed by different amounts of alumina, namely, the chloroform containing ethanol cannot be boiled during air exhaust and fumigation, and the chloroform from which the ethanol is removed can be boiled, wherein the 6 gradient alumina chromatographic columns can meet the requirement of keeping the boiling time for 3min in microbial chloroform fumigation, so that the effect of killing microorganisms can be achieved, and therefore, the ethanol in the chloroform is extracted with minimum use amount of alumina, namely the ratio of the alumina to the chloroform is 5 g: 100ml of chloroform was prepared.
Example 2
The chloroform without ethanol is used for measuring the soil microorganism amount of the Pinus sylvestris artificial forest:
(1) preparation of soil samples
The experiment of this example utilized 6 fresh soil samples of 0-10cm taken from the pinus sylvestris forest, sample numbers 1, 2, 3, 4, 5, 6. 12 parts of 20g fresh soil is weighed into a 50ml glass beaker for each soil sample, wherein 3 parts are used for fumigating ethanol-free chloroform after being treated by the method of the invention, 3 parts are used for fumigating with commercial ethanol-containing chloroform as a control, 3 parts are used for fumigating with chloroform prepared by liquid-separating distillation recorded in the background technology as a control, and 3 parts are used as a blank control without fumigating.
(2) Fumigating
25ml of alumina prepared in the above example in a ratio of 5g to chloroform: 100ml of ethanol-free chloroform collected after adsorption is poured into a 50ml beaker, a small spoon of anti-explosive particles is added, and simultaneously the beaker containing the soil to be fumigated and the beaker containing 30ml of 2M sodium hydroxide are put into a vacuum drier together. Sealing the dryer with a small amount of vaseline, pumping with an air pump until chloroform boils for 3min, closing the valve of the dryer, and fumigating at 25 deg.C in dark for 3 days. Chloroform was distilled by liquid separation in the same manner, and a soil sample was fumigated with commercially available chloroform containing ethanol.
After the end of fumigation, the desiccator was opened, the beaker containing ethanol-free chloroform was taken out, the remaining chloroform was poured into a brown reagent bottle, and 5g of anhydrous potassium carbonate was added for preservation. After the chloroform was removed, the desiccator was evacuated several more times to remove the residual chloroform from the soil. Taking out the soil sample, leaching with 0..5M potassium sulfate, and measuring the content of active carbon in the leaching solution.
(3) And (5) measuring soil activated carbon.
Accurately sucking 5ml of the leaching solution into a 18 x 180 test tube, adding 5ml of potassium dichromate solution, adding a small spoon of zeolite, slowly heating in an oil bath kettle at 170 ℃, starting boiling for 10min, taking out, slightly cooling, wiping off oil stains on the wall of the test tube, transferring a small amount of distilled water to a triangular flask without loss for many times, adding 2 drops of an o-phenanthroline indicator, titrating with a ferrous sulfate solution, and taking the solution from orange → blue-green → brick red as a titration end point and simultaneously making a blank and calibrating the concentration of ferrous sulfate.
(4) Microbial biomass carbon calculation
And calculating the difference value of the activated carbon in the fumigated soil and the non-fumigated soil, and dividing the difference value by a corresponding coefficient to obtain the soil microbial biomass carbon (see tables 1, 2 and 3).
W(c)＝Ec/KEc
In the formula:
w (c) -carbon mass fraction of microorganisms (mg. kg)-1)
Ec-difference between fumigated and non-fumigated
KEc-extraction coefficient (0.38 is taken)
TABLE 1 carbon concentration of chloroform fumigation treatment soil microorganisms prepared according to the present invention
TABLE 2 preparation of carbon concentration of chloroform fumigated soil microorganism by liquid separation distillation
TABLE 3 carbon concentration of commercial ethanol-containing chloroform fumigated soil microorganisms
As a result: as can be seen from tables 1, 2 and 3, the carbon content of the soil microorganisms of the commercial ethanol-containing chloroform fumigation treatment is significantly lower than that of the soil microorganisms of the other 2 fumigation treatments. The carbon content of the soil microorganism treated by chloroform fumigation and the carbon content of the soil microorganism treated by chloroform prepared by liquid-separating distillation have a correlation coefficient R20.9811, r 0.99, reaching a very significant level of 1% (n 6). The slope of the trend line was 0.9834, approaching 1, and the results were essentially consistent for both fumigation treatments. However, the carbon content of the soil microorganisms fumigated and treated by the chloroform prepared by the method is higher than that of the soil microorganisms treated by the chloroform prepared by liquid separation distillation, becauseThe chloroform fumigated soil prepared by the method has better effect of killing microorganisms; in addition, the invention has the advantages of simple equipment and reagent required by the experiment, short required time and greatly reduced toxicity in the preparation process.
Claims (6)
1. A method for rapidly removing ethanol in chloroform is characterized in that: standing and adsorbing chloroform to be treated by alumina particles; thus obtaining chloroform with ethanol removed.
2. The method for rapidly removing ethanol from chloroform as claimed in claim 1, wherein: the volume mass ratio of the chloroform to the alumina particles is 5-30: 100.
3. The method for rapidly removing ethanol from chloroform as claimed in claim 2, wherein: the alumina particles were analytical grade, 100-200 mesh.
4. A method for the rapid removal of ethanol from chloroform as claimed in any one of claims 1 to 3, wherein: injecting the chloroform to be treated into a chromatographic column filled with alumina particles, and standing for 30-40min after injection; then the concentration is controlled to be 20-25 ml.min-1The flow rate of the chloroform was measured and the effluent was collected, i.e., the chloroform from which the ethanol was removed.
5. The method for rapidly removing ethanol from chloroform as claimed in claim 4, wherein: the chromatographic column is filled with an alumina particle layer, and the upper surface and the lower surface of the particle layer are covered with filter paper.
6. The method for rapidly removing ethanol from chloroform as claimed in claim 4, wherein: the filter paper was wetted with chloroform.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CH605494A5 (en) * | 1974-03-26 | 1978-09-29 | Ghirga Marcello | Chloroform or methylene chloride separation from alcohol |
| US5131985A (en) * | 1991-06-03 | 1992-07-21 | Lloyd Berg | Separation of chloroform from a lower alcohol by extractive distillation |
| CN102093159A (en) * | 2010-12-29 | 2011-06-15 | 天津市康科德科技有限公司 | Method for preparing chromatographic pure dichloromethane |
| CN104402666A (en) * | 2014-10-20 | 2015-03-11 | 太仓沪试试剂有限公司 | Purification method for MOS-grade chloroform |
-
2019
- 2019-07-25 CN CN201910674972.1A patent/CN112284841A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CH605494A5 (en) * | 1974-03-26 | 1978-09-29 | Ghirga Marcello | Chloroform or methylene chloride separation from alcohol |
| US5131985A (en) * | 1991-06-03 | 1992-07-21 | Lloyd Berg | Separation of chloroform from a lower alcohol by extractive distillation |
| CN102093159A (en) * | 2010-12-29 | 2011-06-15 | 天津市康科德科技有限公司 | Method for preparing chromatographic pure dichloromethane |
| CN104402666A (en) * | 2014-10-20 | 2015-03-11 | 太仓沪试试剂有限公司 | Purification method for MOS-grade chloroform |
Non-Patent Citations (4)
| Title |
|---|
| W. M. WILLIAMSON ET AL.: "METHOD FOR RAPID REMOVAL OF ETHANOL FROM CHLOROFORM IN SOIL MICROBIAL BIOMASS DETERMINATIONS", 《COMMUN. SOIL SCI. PLANT ANAL.》 * |
| 周桦等: "氯仿薰蒸浸提法测定土壤微生物量碳的改进", 《土壤通报》 * |
| 唐嗣荣: "吸附法生产无水乙醇的技术进展", 《化工设计》 * |
| 穆光照等: "《实用溶剂手册》", 30 September 1990 * |
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Application publication date: 20210129 |