CN116355698A - Method for safely and efficiently extracting soil phospholipid fatty acid by using dichloromethane - Google Patents
Method for safely and efficiently extracting soil phospholipid fatty acid by using dichloromethane Download PDFInfo
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- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 title claims abstract description 185
- 239000002689 soil Substances 0.000 title claims abstract description 56
- 235000014113 dietary fatty acids Nutrition 0.000 title claims abstract description 46
- 239000000194 fatty acid Substances 0.000 title claims abstract description 46
- 229930195729 fatty acid Natural products 0.000 title claims abstract description 46
- -1 phospholipid fatty acid Chemical class 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 30
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 69
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 51
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 36
- 239000000243 solution Substances 0.000 claims description 33
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 24
- 238000000605 extraction Methods 0.000 claims description 23
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 238000002386 leaching Methods 0.000 claims description 19
- 150000003904 phospholipids Chemical class 0.000 claims description 18
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 229910052757 nitrogen Inorganic materials 0.000 claims description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 11
- 239000011259 mixed solution Substances 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 9
- BDXAHSJUDUZLDU-UHFFFAOYSA-N methyl nonadecanoate Chemical compound CCCCCCCCCCCCCCCCCCC(=O)OC BDXAHSJUDUZLDU-UHFFFAOYSA-N 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 5
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 239000012074 organic phase Substances 0.000 claims description 5
- 230000010355 oscillation Effects 0.000 claims description 5
- CASUWPDYGGAUQV-UHFFFAOYSA-M potassium;methanol;hydroxide Chemical compound [OH-].[K+].OC CASUWPDYGGAUQV-UHFFFAOYSA-M 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 239000001509 sodium citrate Substances 0.000 claims description 4
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 4
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 claims description 3
- 229930186217 Glycolipid Natural products 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 abstract description 42
- 125000001309 chloro group Chemical group Cl* 0.000 abstract description 3
- 231100001231 less toxic Toxicity 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 10
- 238000012360 testing method Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 6
- 238000000926 separation method Methods 0.000 description 4
- 229960000583 acetic acid Drugs 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000003260 vortexing Methods 0.000 description 3
- 238000009835 boiling Methods 0.000 description 2
- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical compound ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- QKGYJVXSKCDGOK-UHFFFAOYSA-N hexane;propan-2-ol Chemical compound CC(C)O.CCCCCC QKGYJVXSKCDGOK-UHFFFAOYSA-N 0.000 description 1
- NGISWXJYJXIYEF-UHFFFAOYSA-N hexane;propan-2-ol;hydrate Chemical compound O.CC(C)O.CCCCCC NGISWXJYJXIYEF-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 231100000003 human carcinogen Toxicity 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- ZOCYQVNGROEVLU-UHFFFAOYSA-N isopentadecanoic acid Chemical compound CC(C)CCCCCCCCCCCC(O)=O ZOCYQVNGROEVLU-UHFFFAOYSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- BKBMACKZOSMMGT-UHFFFAOYSA-N methanol;toluene Chemical compound OC.CC1=CC=CC=C1 BKBMACKZOSMMGT-UHFFFAOYSA-N 0.000 description 1
- 229940050176 methyl chloride Drugs 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
- C11C1/00—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
- C11C1/002—Sources of fatty acids, e.g. natural glycerides, characterised by the nature, the quantities or the distribution of said acids
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
- C11C1/00—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
- C11C1/007—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids using organic solvents
-
- 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
- Y02W30/74—Recovery of fats, fatty oils, fatty acids or other fatty substances, e.g. lanolin or waxes
Abstract
The invention belongs to the technical field of biosafety, and relates to a method for safely and efficiently extracting soil phospholipid fatty acid by using methylene dichloride. The invention utilizes the characteristics that the dichloromethane is more stable and less toxic than the similar chloro compounds, and the dichloromethane is used for replacing the conventional chloroform, so that the efficiency of extracting the soil phospholipid fatty acid (PLFA) is greatly improved, and the method is healthier, safer and more friendly to human bodies and the environment.
Description
Technical Field
The invention belongs to the technical field of biosafety, and relates to a method for safely and efficiently extracting soil phospholipid fatty acid by using methylene dichloride.
Background
Traditional methods for extracting phospholipid fatty acids (PLFA) from soil use chloroform: methanol: citrate = 1:2: extraction at a ratio of 0.8 is widely used because of its many advantages. Chloroform, however, has been listed as a possible human carcinogen, with damage to the heart, liver, kidneys. In many cases, the use of chloroform has been restricted or prohibited. Therefore, it is imperative to develop a safer organic solvent to replace chloroform to reduce the harm to human body.
The nature of dichloromethane and chloroform and their similarity, are generally interchangeable. For specific substances, the solubility of the two substances is different, the boiling points of the two substances are different greatly, and the polarity of chloroform is large, but in the extraction of the soil phospholipid fatty acid (PLFA), the chloroform only acts as an extractant, and the solubility and the boiling point have no influence on the test result. Although chloroform with large polarity can extract more fatty acid carbon chains, for most of the soil, the use of dichloromethane is completely enough to extract phospholipids in the soil, and has no influence on analysis of experimental results. If it is relatively barren, chloroform extraction may be suitably selected.
Chloroform has great harm to human body, and the treatment of chloroform waste liquid after experiments is also very careful, otherwise, the environment is greatly polluted. Dichloromethane is more stable than the similar chloro compounds, and is the least toxic of methyl chloride. Extraction of the soil phospholipid fatty acids (PLFA) using methylene chloride instead of chloroform would be a more healthy, safe and friendly method for the human body and environment.
Disclosure of Invention
The invention aims at solving the problems in the prior art and is a method for safely and efficiently extracting the phospholipid fatty acid in the soil by using methylene dichloride, which is healthier for human bodies and the environment
The aim of the invention can be achieved by the following technical scheme: a method of extracting a soil phospholipid fatty acid, the method comprising the steps of:
s1, sequentially adding a citric acid buffer solution, dichloromethane and methanol into soil for leaching to obtain a leaching solution;
s2, centrifugally separating the leaching solution, repeating the step S1 to carry out secondary leaching on the centrifuged bottom soil sample, then sequentially adding n-hexane, isopropanol and water to carry out tertiary leaching, and combining leaching solutions of the tertiary leaching;
s3, adding a citric acid buffer solution and methylene dichloride into the leaching solution for shading and standing, taking a methylene dichloride solution at the lower layer after standing and layering, adding methanol for oscillation, drying by nitrogen, and extracting by an SPE extraction column to obtain a phospholipid solution;
s4, adding a methanol/toluene mixed solution into the phospholipid solution for dissolution, then adding a potassium hydroxide methanol solution for water bath heating, then adding n-hexane/dichloromethane mixed solution, adding acetic acid and deionized water for oscillating centrifugation, finally adding n-hexane/isopropanol for oscillating centrifugation, transferring an upper organic phase, and drying by nitrogen.
In the method for extracting the soil phospholipid fatty acid, the step S1 citric acid buffer solution is prepared by mixing a sodium citrate aqueous solution with the pH of 4, specifically 0.1-0.2moL/L and a citric acid aqueous solution with the pH of 0.1-0.2 moL/L.
In the method for extracting the soil phospholipid fatty acid, the dichloromethane contains 5-10% (w/v) of ethanol. In order to ensure higher fatty acid extraction efficiency, the invention adds 5-10% (w/v) ethanol to prevent loss of phospholipid fatty acid, and can improve the extraction efficiency of extracting the phospholipid fatty acid in soil.
In the above method for extracting the soil phospholipid fatty acid, the volume ratio of the citric acid buffer solution, the dichloromethane and the methanol in the step S1 is 1: (1-1.5): (2-3).
Preferably, the volume ratio of n-hexane, isopropanol and water in the step S2 is (5-7): (7-9): 1.5.
in the method for extracting the soil phospholipid fatty acid, the shading and standing time in the step S3 is 20-24 hours.
In the above method for extracting a soil phospholipid fatty acid, the step S3 extraction specifically comprises: the SPE column is washed with dichloromethane, the dichloromethane solution is transferred to the SPE column, then the neutral fat is washed with dichloromethane, the glycolipid is washed with acetone, the phospholipid is washed with methanol, and the internal standard is added, and then the nitrogen is dried.
In the method for extracting the soil phospholipid fatty acid, the internal standard is a solution of methyl nonadecanoate with the concentration of 30-35 ppm. Microorganisms in nature do not contain 19:0 phospholipids, so a solution of methyl nonadecanoate at a concentration of 30-35ppm is used as an internal standard.
In the method for extracting the soil phospholipid fatty acid, the volume ratio of the methanol to the toluene in the methanol/toluene mixed solution in the step S4 is (0.8-1.2): 1.
In the method for extracting the soil phospholipid fatty acid, the concentration of the potassium hydroxide methanol solution in the step S4 is 0.1-0.3moL/L.
In the method for extracting the soil phospholipid fatty acid, the volume ratio of the n-hexane to the dichloromethane in the n-hexane/dichloromethane mixed solution in the step S4 is (2-5): 1.
compared with the prior art, the invention has the following beneficial effects: the invention utilizes the characteristics that the dichloromethane is more stable and less toxic than the similar chloro compounds, and the dichloromethane is used for replacing the conventional chloroform, so that the efficiency of extracting the soil phospholipid fatty acid (PLFA) is greatly improved, and the method is healthier, safer and more friendly to human bodies and the environment.
Drawings
FIG. 1 is a graph showing comparison of the response values of phospholipid fatty acid carbon chains in the extracted soil of example 1 and comparative example 1.
FIG. 2 is a graph showing comparison of the concentration of PLFAs in phospholipid fatty acids extracted from soil in example 1 and comparative example 2.
FIG. 3 is a graph showing comparison of the concentration of PLFAs in phospholipid fatty acids extracted from soil in example 1 and comparative example 3.
Description of the embodiments
The following are specific examples of the present invention, and the technical solutions of the present invention are further described, but the present invention is not limited to these examples.
Citric acid buffer at a concentration of 0.15 moL/L: 100mL of 0.15moL/L sodium citrate (4.41 g sodium citrate in 100mL deionized water) and 150mL of 0.15moL/L citric acid solution (6.3 g citric acid in 200 mL deionized water) were mixed and the pH was adjusted to 4 with citric acid solution.
Internal standard: 100ml of the internal standard solution with the concentration of 31ppm is prepared by weighing 0.003100099887g of an internal standard pure product (methyl nonadecanoate) and dissolving the product in 100ml of n-hexane.
N-hexane/dichloromethane mixture: 60mL of n-hexane, 15mL of methylene chloride.
Methanol toluene mixed solution: the volume ratio is 1:1.
Potassium hydroxide methanol solution at 0.2 moL/L: 0.56g KOH was dissolved in 50mL methanol.
Glacial acetic acid at concentration 1M: 2.9mL was dissolved in 50mL of water.
Dichloromethane: ethanol stabilized methylene chloride was formulated by distilling HPLC grade methylene chloride to remove other impurities and then adding 8% (w/v) ethanol.
Examples
Day1: weighing 1.5g of freeze-dried acidic soil sample into a 50mL glass centrifuge tube, sequentially adding 1.6mL of citric acid buffer solution, 2mL of dichloromethane, 4mL of methanol and carrying out vortex oscillation; the mixture was shaken horizontally under shade for 2 hours (250 rpm), and centrifuged (10 min,1500 rpm). Transfer the solution to a large tube, add 0.8mL of citric acid buffer, 1mL of dichloromethane, 2mL of methanol, vortex shaking, shake horizontally under shade for 2h (250 rpm), centrifuge (10 min,1500 rpm), complete the second leaching, and pour the two leaches into the same large tube. 3mL of normal hexane, 4mL of isopropanol and 0.75mL of water are added into the soil leached twice, vortex oscillation is carried out, horizontal oscillation is carried out for 2 hours (250 rpm) under the condition of shading, centrifugation (10 min,1500 rpm) is carried out, eluent is poured into the same large test tube, and complete elution is carried out on phospholipid fatty acid in the soil, so that complete separation of phospholipid is realized. Add 2.4mL of citric acid buffer and 3mL of dichloromethane to the large tube of the dump leaching solution. The lid was closed, shaken for 1min, and deflated at intervals. Shading and standing for 20h (4 ℃ C. Refrigerator preservation).
Day2: the lower methylene chloride phase was transferred to a medium glass tube (care was taken not to aspirate the upper solution during transfer, the nitrogen was blown dry (temperature was not to exceed 40 ℃ C., and was typically set to 25-30 ℃ C.) and 1mL of methanol was added, vortexing was performed to allow sufficient dissolution, nitrogen was blown dry, 1mL of methanol was repeatedly added, vortexing was performed and nitrogen was blown dry, SPE columns were prepared, 5mL of methylene chloride was washed, the sample was put on the column, 1mL of methylene chloride was used to dissolve the sample and transferred to the SPE column, repeating twice, 10mL of methylene chloride was added to wash neutral fat, 10mL of acetone was added to wash glycolipid, the waste liquid in the SPE extraction apparatus was poured off, the tube holder with test tube was placed in the SPE extraction apparatus, 8mL of methanol was added, the washed phospholipid was put in a small tube, and the collection was blown dry with nitrogen, 200. Mu.L of internal standard (vortexing), nitrogen was added, and stored at zero.
Day3: 1mL of a methanol/toluene mixture was added to dissolve the phospholipid, 1mL of a 0.2 mL/L potassium hydroxide methanol solution was added to conduct a water bath at 37℃for 15min, and 2mL of a n-hexane/dichloromethane mixture was added. 0.3mL of 1M acetic acid was added, 2mL of deionized water was added, the mixture was separated by shaking (10 min), centrifuged (1500 rpm,5 min), and the upper organic phase was transferred to a small-size tube.
Wherein 2mL of a mixture of n-hexane and methylene chloride was added to the original test tube for repeated extraction, shaking separation (10 min), centrifugation (1500 rpm,5 min), transferring the upper organic phase to the same small test tube, adding 1.5mL of n-hexane, 2mL of isopropyl alcohol to the original test tube for complete extraction again, shaking separation (10 min), centrifugation (1500 rpm,5 min), transferring the upper organic phase to the same small test tube. Finally, drying with nitrogen.
And (3) measuring by an upper machine: the phospholipid fatty acid methyl ester was dissolved 75 microliters (2 times) with n-hexane (with a gun), transferred into the GC inner cannula, and put on-line.
Examples
The difference from example 1 is only that no ethanol was added to the methylene chloride.
Examples
The difference from example 1 is only that 1% (w/v) ethanol was added to methylene chloride.
Examples
The difference from example 1 is only that 1.5g of the freeze-dried soil sample was weighed as an alkaline soil sample.
Comparative example 1:
and extracting the phospholipid fatty acid carbon chains in the soil by using a conventional chloroform extraction method.
Comparative example 2:
the difference from example 1 is only that ethanol-stabilized methylene chloride was replaced by chloroform.
Comparative example 3:
the difference from example 4 is only that ethanol-stabilized methylene chloride was replaced by chloroform.
Table 1: examples 1-3 and comparative examples 1-2 extraction of phospholipid fatty acid carbon chains
FIG. 1 is a graph showing comparison of the response values of phospholipid fatty acid carbon chains in the extracted soil of example 1 and comparative example 1. As can be seen from the graph, the response values of 15:0 iso,15:1 w6c,15:0 DMA,16:0 iso,16:0 anteiso,16:1 w9c,16:1 w7c,16:1 w5c,16:0 10-methyl,17:0 iso,17:0 anteiso,17:1 w8c,17:0 cyclo w7c,17:0, 17:1 w7c 10-methyl,17:0 10-methyl,17:0 DMA,18:1 w7c,18:1 w7c 10-methyl,18:0 10-methyl,19:1 w8c,19:0 cyclo w7c,20:4 w6c,20:5 w3c,20:1 w9c,21:1 w8c,21:1 w3c,23:1 w4c and the like of the invention are obviously increased compared with the response values of chloroform extraction, and the extraction effect is obviously better.
FIG. 2 is a graph showing comparison of the concentration of PLFAs in phospholipid fatty acids extracted from soil in example 1 and comparative example 2. From the figure, the concentrations of fatty acid carbon chains of 16:0, 18:0, 20:0, 14:0, a15:0, i15:0, i16:0, 17:0, i17:0, 10Me17:0, 10Me18:0, 16:1ω7, cy17:0, cy19:0, 18:1ω9c,18:1ω9t,18:2ω6,9c,23:0, 24:0, etc. were significantly higher than those of conventional chloroform extraction under modified conditions. It is explained that extraction of phospholipids in soil with ethanol stabilized methylene chloride can result in more complete leaching of the phospholipid components in soil.
FIG. 3 is a graph showing comparison of the concentration of PLFAs in phospholipid fatty acids extracted from soil in example 1 and comparative example 3. From the graph, the results of the alkaline soil are basically consistent with those of the acid soil, and the concentrations of fatty acid carbon chains such as 16:0, 18:0, 20:0, 14:0, 15:0, a15:0, i15:0, i16:0, 10Me16:0, 10Me17:0, 16:1ω7, cy17:0, cy19:0, 18:1ω9t,23:0, 24:0 and the like can be obviously higher than those of the conventional chloroform extraction under the improved condition. The invention is demonstrated that the extraction of the phospholipid in the soil by using the ethanol-stabilized methylene dichloride can lead the phospholipid component in the soil to be leached more completely.
In summary, the invention uses the methylene dichloride which is added with stable ethanol to replace chloroform for extraction, which not only has little harm to human bodies and environment, but also prevents the loss of phospholipid fatty acid by adding 5-10% (w/v) ethanol, and the invention can realize the complete separation of the phospholipid component of the soil by respectively adding n-hexane-isopropanol-water (6:8:1.5) and n-hexane-isopropanol (3:4) for elution in the traditional steps of Day1 and Day3, thereby achieving more efficient extraction.
The point values in the technical scope of the present invention are not exhaustive, and the new technical solutions formed by equivalent substitution of single or multiple technical features in the technical solutions of the embodiments are also within the scope of the present invention; meanwhile, in all the listed or unrecited embodiments of the present invention, each parameter in the same embodiment represents only one example of the technical scheme (i.e. a feasibility scheme), and no strict coordination and limitation relation exists between each parameter, wherein each parameter can be replaced with each other without violating axiom and the requirement of the present invention, except what is specifically stated.
The technical means disclosed by the scheme of the invention is not limited to the technical means disclosed by the technical means, and also comprises the technical scheme formed by any combination of the technical features. While the foregoing is directed to embodiments of the present invention, it will be appreciated by those skilled in the art that changes and modifications may be made without departing from the principles of the invention, and such changes and modifications are intended to be included within the scope of the invention.
The specific embodiments described herein are offered by way of example only to illustrate the spirit of the invention. Those skilled in the art may make various modifications or additions to the described embodiments or substitutions thereof without departing from the spirit of the invention or exceeding the scope of the invention as defined in the accompanying claims.
Claims (10)
1. A method for safely and efficiently extracting soil phospholipid fatty acid by using methylene dichloride, which is characterized by comprising the following steps of:
s1, sequentially adding a citric acid buffer solution, dichloromethane and methanol into soil for leaching to obtain a leaching solution,
s2, centrifugally separating the leaching solution, repeating the step S1 to carry out secondary leaching on the centrifuged bottom soil sample, then sequentially adding n-hexane, isopropanol and water to carry out tertiary leaching, and combining leaching solutions of the tertiary leaching;
s3, adding a citric acid buffer solution and methylene dichloride into the leaching solution for shading and standing, taking a methylene dichloride solution at the lower layer after standing and layering, adding methanol for oscillation, drying by nitrogen, and extracting by an SPE extraction column to obtain a phospholipid solution;
s4, adding a methanol/toluene mixed solution into the phospholipid solution for dissolution, then adding a potassium hydroxide methanol solution for water bath heating, then adding an n-hexane/dichloromethane mixed solution, adding acetic acid and deionized water for oscillating centrifugation, finally adding an n-hexane/isopropanol mixed solution for oscillating centrifugation, transferring an upper organic phase, and drying by nitrogen.
2. The method for safely and efficiently extracting the phospholipid fatty acids in the soil by using methylene chloride according to claim 1, wherein the citric acid buffer solution in the step S1 is prepared by mixing a sodium citrate aqueous solution with the pH of 4, specifically 0.1-0.2moL/L and a citric acid aqueous solution with the pH of 0.1-0.2 moL/L.
3. The method for safely and efficiently extracting a phospholipid fatty acid from soil using methylene chloride according to claim 1, wherein the methylene chloride contains 5-10% (w/v) ethanol.
4. The method for safely and efficiently extracting the soil phospholipid fatty acid by using the methylene chloride according to claim 1, wherein the volume ratio of the citric acid buffer solution to the methylene chloride to the methanol in the step S1 is 1: (1-1.5): (2-3).
5. The method for safely and efficiently extracting the phospholipid fatty acids in the soil by using methylene chloride according to claim 1, wherein the shading and standing time in the step S3 is 20-24 hours.
6. The method for safely and efficiently extracting the phospholipid fatty acids in the soil by using the methylene chloride according to claim 1, wherein the step S3 is specifically extracted as follows: the SPE column is washed with dichloromethane, the dichloromethane solution is transferred to the SPE column, then the neutral fat is washed with dichloromethane, the glycolipid is washed with acetone, the phospholipid is washed with methanol, and the internal standard is added, and then the nitrogen is dried.
7. The method for safely and efficiently extracting a soil phospholipid fatty acid with dichloromethane according to claim 5, wherein the internal standard is a solution of methyl nonadecanoate with a concentration of 30-35 ppm.
8. The method for safely and efficiently extracting the soil phospholipid fatty acid by using the methylene chloride according to claim 1, wherein the volume ratio of methanol to toluene in the methanol/toluene mixed solution in the step S4 is (0.8-1.2): 1.
9. The method for safely and efficiently extracting a soil phospholipid fatty acid by using methylene chloride according to claim 1, wherein the concentration of the methanol solution of potassium hydroxide in the step S4 is 0.1-0.3moL/L.
10. The method for safely and efficiently extracting the soil phospholipid fatty acid by using the methylene dichloride according to claim 1, wherein the volume ratio of the n-hexane to the methylene dichloride in the mixed solution of n-hexane and methylene dichloride in the step S4 is (2-5): 1.
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