Method for extracting n-alkanes from dianthus chinensis

Abstract

The invention discloses a method for extracting a lipid biomarker, namely n-alkanes, from stalagmite. The method comprises the following steps: 1) digesting a stalagmite sample with acid; 2) carrying out microwave-assisted digestion at a certain temperature; 3) extracting a lipid extract; 4) concentrating the lipid extract; by changing the traditional reagent and extraction steps and combining the physicochemical characteristics of the kyllite, the biomarker is creatively released from the kyllite, and subsequent extraction is performed, so that the problem of low efficiency of extracting the lipid biomarker-n-alkane from the kyllite at present is solved. The invention belongs to the technical field of natural product extraction and separation.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction and separation, and particularly relates to a method for extracting lipid biomarkers, namely normal alkanes, from stalagmites. Background Art

[0002] n-Alkanes consist of a series of adjacent methyl groups with their carbon atoms arranged in straight chains and are generally biosynthetic in nature. They are mainly derived from leaf waxes of terrestrial plants in nature. Leaf wax is a thin waxy covering on the plant epidermis that is synthesized and secreted by the plant. n-Alkanes are widely found in modern plants, soils, peatlands, stalagmites, river sediments, and lake and marine sediments. Because n-alkanes are straight-chain hydrocarbons and lack reactive functional groups, they are very stable and environmentally persistent, remaining in the fossil record for tens of millions of years.

[0003] Carbonate sediments in caves, especially stalagmites, record carbonate accumulation over long time scales and have great potential to provide high-resolution paleoclimate records. In addition to the inorganic indicators in stalagmites that have been widely used (carbonate carbon isotopes, oxygen isotopes, trace elements, etc.), low-abundance organic matter preserved in stalagmites also records important environmental information. Among them, n-alkanes are very sensitive to environmental changes. For example, n-alkanes with a certain carbon number range, different distribution types, dominant carbon numbers and different indicators (such as average chain length (ACL), carbon preference index (CPI), etc.) can record biogenic information. Therefore, proxy indicators based on the distribution of n-alkane molecules have been widely used to reconstruct paleoenvironmental changes.

[0004] However, stalagmite samples are very precious, and the content of n-alkanes preserved in them is extremely low. When n-alkanes exist in a limestone environment, they are closely combined with calcite or other components through physical and chemical reactions, which enhances the stability of organic matter in calcite. This makes it difficult for n-alkanes to be released from calcite through simple dissolution, extraction or digestion processes. The existing method of extracting n-alkanes from stalagmites mainly uses the method of directly using acid to digest calcite and then extract it. The extraction efficiency is low, and it is difficult to extract enough n-alkanes for detection, resulting in a waste of precious stalagmite raw materials. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the object of the present invention is to provide a method for extracting normal alkanes from stalagmites, using microwave digestion to act on a stalagmite digestion solution to achieve the purpose of releasing more normal alkanes from calcite crystals and extracting more normal alkanes with less stalagmite.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a method for extracting normal alkanes from stalagmites, which specifically comprises the following steps: Step 1, taking a stalagmite sample, grinding it and performing acid hydrolysis to form a stalagmite digestion solution; Step 2, heating the stalagmite digestion solution to 40° C. to 80° C. and keeping the temperature, performing a digestion reaction with microwave assistance, and cooling after the reaction to form a solution to be extracted; Step 3, mixing the liquid to be extracted with the extractant and performing multiple extractions, and concentrating to obtain a solid lipid extract; Step 4, separating the lipid extract by silica gel column chromatography, and extracting the normal alkanes in the stalagmite sample after air drying.

[0007] The working principle of the digestion instrument is to use the thermal effect and non-thermal effect of microwaves to heat and decompose the sample. Microwaves refer to electromagnetic waves with a frequency between 300MHz and 300GHz, which can quickly penetrate the container wall and directly act on the sample and acid mixture, increase the friction between molecules, and thus generate heat. The digestion process can destroy the complex matrix of the sample, extract the target analyte from the complex matrix, and convert it into a form suitable for detection by analytical instruments such as spectroscopy and chromatography. The use of microwave heating to assist in the digestion reaction of stalagmite samples is one of the inventive points of the present invention. The common main use of microwave digestion instruments is to convert solid or insoluble samples into solution form for elemental analysis, heavy metal detection, organic pollutant analysis, etc. This technology uses microwave energy to break the chemical bonds in the crystal, so that more lipids are released into the solution. By using microwave-assisted digestion reactions at 40℃~80℃, the normal alkanes in calcite can be released more thoroughly, thereby increasing the amount of normal alkanes extracted from a unit stalagmite sample.

[0008] Preferably, hydrochloric acid is used to acidify the stalagmite sample in step 1, and the concentration of hydrochloric acid is 2.5~3.5mol / L. For the digestion of calcite, commonly used digestion solutions include strong acids such as hydrochloric acid, nitric acid, and hydrofluoric acid, but considering the characteristics of different extraction targets, it is necessary to select a suitable acidolysis agent. For the extraction of target normal alkanes, it is not advisable to use an acidic solution that causes a too violent digestion reaction for digestion, so as not to destroy the organic matter, and it is not advisable to use a digestion solution with too weak acidity for digestion, otherwise it may cause incomplete digestion, which affects the subsequent extraction steps, such as the effect of microwave digestion.

[0009] Preferably, the mass volume ratio of the stalagmite sample and the hydrochloric acid in step 1 is 3: (18-30) g / mL, so that the blocky stalagmite solid sample completely disappears and no bubbles are generated in this step.

[0010] Further preferably, the heating temperature in step 2 is 60°C to 80°C, with 80°C being the best.

[0011] High temperature helps to break the chemical bonds in the rock, making it easier for organic matter to be decomposed and released. Microwave heating is selective and can preferentially heat the polar molecules in the sample. At the appropriate temperature, this selective heating can more effectively promote the decomposition of organic matter such as lipids. At 0~80℃, the higher the temperature, the better the release of organic matter. When it exceeds 80℃, the target organic matter may decompose, resulting in reduced extraction efficiency.

[0012] Preferably, in step 2, the heating time to the insulation temperature is 3 to 7 minutes, the insulation time is 15 to 25 minutes, and then the mixture is cooled to below 20°C.

[0013] Since the extractants used in the subsequent extraction process, such as organic reagents such as dichloromethane, have very low boiling points and are easily volatile, the extract needs to be cooled to below the boiling point of the extractant after microwave digestion to ensure the extraction process proceeds smoothly.

[0014] Preferably, the extractant in step 3 is dichloromethane, n-hexane, methanol or acetone.

[0015] Preferably, in step 3, the extractant is evaporated to concentrate the lipid extract.

[0016] Preferably, dichloromethane is used as the extractant to extract lipids in a fully automatic liquid-liquid extractor, and the extraction is performed 6 times, each time for 10 minutes.

[0017] The temperature of evaporating the extractant in step 3 is 30-40° C. until the extractant is completely volatilized. Alternatively, the extractant in the lipid extract after extraction can be directly evaporated using an evaporator.

[0018] Preferably, the eluent in step 4 is n-hexane, cyclohexane or petroleum ether.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for extracting normal alkanes from stalagmites, using the thermal effect and non-thermal effect of microwave digestion to heat and decompose the sample, and using microwave-assisted digestion reaction at 40°C to 80°C to destroy the complex matrix of the sample, so that the normal alkanes in the calcite can be released more thoroughly, and at the same time, the target normal alkanes will not be destroyed, thereby increasing the amount of normal alkanes extracted from the unit stalagmites sample. At the optimal temperature of 80°C, the normal alkane extraction efficiency is increased by 57.23% compared with the control group without microwave digestion. The present invention uses the energy generated by microwaves to release more normal alkanes wrapped in calcite, which not only saves stalagmites samples, but also enables the content of extracted normal alkanes to meet the measurement of carbon and hydrogen isotopes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1Figure 2 is a graph of the normal alkane content extracted from various embodiments of the present invention; Figure 2 Flow chart of the extraction steps of the present invention. DETAILED DESCRIPTION

[0021] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0022] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0023] The present invention is further described in detail below in conjunction with the accompanying drawings: Example 1 like Figure 2 As shown, the optimization method for extracting normal alkanes from stalagmites provided by the present invention comprises the following steps: Step 1: take about 6 g of stalagmite sample collected from Furong Cave in Chongqing, my country, use about 40 mL of 3 mol / L hydrochloric acid to completely digest it to form a digestion solution, and place it in a 60 mL glass bottle; Step 2: Place the digestate in an APL Touchwin 4.0 microwave digester, set the temperature to 40°C, the ramp time to 5 minutes, and the hold time to 20 minutes. After the instrument is finished running, wait for the digestate to cool to below 20°C; Step 3, using 20 ml of dichloromethane to extract lipids in a CYZLDZ-6 fully automatic liquid-liquid extractor for 10 min at a time, setting the knob to 1 / 4 during extraction, setting the time to 10 min, and performing 6 extractions for each sample; Step 4: Use the N-1300D-WB rotary evaporator to remove the solvent, and use it with the DLK-2003 low-temperature cooling circulation pump and SB-1300 water bath. Set the operating temperature of the DLK-2003 low-temperature cooling circulation pump to 0°C, and the SB-1300 water bath to maintain a temperature of 36°C to ensure that the dichloromethane can evaporate. Wait until the low-temperature cooling circulation pump cools down to 0°C and the water bath heats up to 36°C before evaporation begins until the liquid is completely evaporated; Step 5, rinse the distillation flask with dichloromethane (5 ml × 3), transfer the lipids to a 20 ml vial, air dry naturally or blow dry with nitrogen, and then use n-hexane solution to further extract the neutral supernatant containing n-alkanes through silica gel column chromatography, and wait for air drying.

[0024] Example 2 like Figure 2 As shown, the optimized method for extracting normal alkanes from stalagmites comprises the following steps: Step 1: take about 6 g of stalagmite sample collected from Furong Cave in Chongqing, my country, use about 40 mL of 3 mol / L hydrochloric acid to completely digest it to form a digestion solution, and place it in a 60 mL glass bottle; Step 2: Place the digestate in an APL Touchwin 4.0 microwave digester, set the temperature to 60°C, the ramp time to 5 minutes, and the hold time to 20 minutes. After the instrument is finished running, wait for the digestate to cool to below 20°C; Step 3, dichloromethane (20 ml × 6) was used to extract lipids in a CYZLDZ-6 fully automatic liquid-liquid extractor for 10 min at a time. During the extraction, the knob was set to 1 / 4 and the time was set to 10 min. Each sample was extracted 6 times; Step 4: Use the N-1300D-WB rotary evaporator to remove the solvent, and use it with the DLK-2003 low-temperature cooling circulation pump and SB-1300 water bath. Set the operating temperature of the DLK-2003 low-temperature cooling circulation pump to 0°C, and the SB-1300 water bath to maintain a temperature of 36°C to ensure that the dichloromethane can evaporate. Wait until the low-temperature cooling circulation pump cools down to 0°C and the water bath heats up to 36°C before evaporation begins until the liquid is completely evaporated; Step 5, rinse the distillation flask with dichloromethane (5 ml × 3), transfer the lipids to a 20 ml vial, air dry naturally or blow dry with nitrogen, and further extract the neutral supernatant containing normal alkanes using n-hexane solution through silica gel column chromatography, and wait for air drying.

[0025] Example 3 like Figure 2 As shown, the optimized method for extracting normal alkanes from stalagmites comprises the following steps: Step 1: take about 6 g of stalagmite sample collected from Furong Cave in Chongqing, my country, use about 40 mL of 3 mol / L hydrochloric acid to completely digest it to form a digestion solution, and place it in a 60 mL glass bottle; Step 2: Place the digestate in an APL Touchwin 4.0 microwave digester, set the holding temperature to 80°C, the ramp time to 5 minutes, and the holding time to 20 minutes. After the instrument is finished running, wait for the digestate to cool to below 20°C; Step 3, dichloromethane (20 ml × 6) was used to extract lipids in a CYZLDZ-6 fully automatic liquid-liquid extractor for 10 min at a time. During the extraction, the knob was set to 1 / 4 and the time was set to 10 min. Each sample was extracted 6 times; Step 4: Use the N-1300D-WB rotary evaporator to remove the solvent, and use it with the DLK-2003 low-temperature cooling circulation pump and SB-1300 water bath. Set the operating temperature of the DLK-2003 low-temperature cooling circulation pump to 0°C, and the SB-1300 water bath to maintain a temperature of 36°C to ensure that the dichloromethane can evaporate. Wait until the low-temperature cooling circulation pump cools down to 0°C and the water bath heats up to 36°C before evaporation begins until the liquid is completely evaporated; Step 5, rinse the distillation flask with dichloromethane (5 ml × 3), transfer the lipids to a 20 ml vial, air dry naturally or blow dry with nitrogen, and further extract the neutral supernatant containing normal alkanes using n-hexane solution through silica gel column chromatography, and wait for air drying.

[0026] Example 4 like Figure 2 As shown, the optimization method for extracting normal alkanes from stalagmites provided by the present invention specifically comprises the following steps: Step 1: take about 6 g of stalagmite sample collected from Furong Cave in Chongqing, my country, use about 40 mL of 3 mol / L hydrochloric acid to completely digest it to form a digestion solution, and place it in a 60 mL glass bottle; Step 2: Place the digestate in an APL Touchwin 4.0 microwave digester, set the holding temperature to 100°C, the ramp time to 5 minutes, and the holding time to 20 minutes. After the instrument is finished running, wait for the digestate to cool to below 20°C; Step 3, dichloromethane (20 ml × 6) was used to extract lipids in a CYZLDZ-6 fully automatic liquid-liquid extractor for 10 min at a time. During the extraction, the knob was set to 1 / 4 and the time was set to 10 min. Each sample was extracted 6 times; Step 4: Use the N-1300D-WB rotary evaporator to remove the solvent, and use it with the DLK-2003 low-temperature cooling circulation pump and SB-1300 water bath. Set the DLK-2003 low-temperature cooling circulation pump to work at 0°C, and the SB-1300 water bath to maintain a temperature of 36°C to ensure that the dichloromethane can evaporate until the liquid is completely evaporated. Wait until the low-temperature cooling circulation pump cools down to 0°C and the water bath heats up to 36°C before evaporation begins; Step 5, rinse the distillation flask with dichloromethane (5 ml × 3), transfer the lipids to a 20 ml vial, air dry naturally or blow dry with nitrogen, and further extract the neutral supernatant containing normal alkanes using n-hexane solution through silica gel column chromatography, and wait for air drying.

[0027] Example 5 like Figure 2 As shown, the optimization method for extracting normal alkanes from stalagmites provided by the present invention comprises the following steps: Step 1: take about 6 g of stalagmite sample collected from Furong Cave in Chongqing, my country, use about 40 mL of 3 mol / L hydrochloric acid to completely digest it to form a digestion solution, and place it in a 60 mL glass bottle; Step 2: Place the digestate in an APL Touchwin 4.0 microwave digester, set the holding temperature to 120°C, the ramp time to 5 minutes, and the holding time to 20 minutes. After the instrument is finished running, wait for the digestate to cool to below 20°C; Step 3, dichloromethane (20 ml × 6) was used to extract lipids in a CYZLDZ-6 fully automatic liquid-liquid extractor for 10 min at a time. During the extraction, the knob was set to 1 / 4 and the time was set to 10 min. Each sample was extracted 6 times; Step 4: Use the N-1300D-WB rotary evaporator to remove the solvent, and use it with the DLK-2003 low-temperature cooling circulation pump and SB-1300 water bath. Set the DLK-2003 low-temperature cooling circulation pump to work at 0°C, and the SB-1300 water bath to maintain a temperature of 36°C to ensure that the dichloromethane can evaporate until the liquid is completely evaporated. Wait until the low-temperature cooling circulation pump cools down to 0°C and the water bath heats up to 36°C before evaporation begins; Step 5, rinse the distillation flask with dichloromethane (5 ml × 3), transfer the lipids to a 20 ml vial, air dry naturally or blow dry with nitrogen, and further extract the neutral supernatant containing normal alkanes using n-hexane solution through silica gel column chromatography, and wait for air drying.

[0028] Comparative Example 1 The optimized method for extracting normal alkanes from stalagmites provided by the present invention comprises the following steps: Step 1: take about 6 g of stalagmite sample collected from Furong Cave in Chongqing, my country, use about 40 mL of 3 mol / L hydrochloric acid to completely digest it to form a digestion solution, and place it in a 60 mL glass bottle; Step 2, dichloromethane (20 ml × 6) was used to extract lipids in a CYZLDZ-6 fully automatic liquid-liquid extractor for 10 min at a time. During the extraction, the knob was set to 1 / 4 and the time was set to 10 min. Each sample was extracted 6 times; Step 3, use the N-1300D-WB rotary evaporator to remove the solvent, and use it with the DLK-2003 low-temperature cooling circulation pump and SB-1300 water bath. Set the DLK-2003 low-temperature cooling circulation pump to work at 0°C, and the SB-1300 water bath to maintain a temperature of 36°C to ensure that the dichloromethane can evaporate until the liquid is completely evaporated. Wait for the low-temperature cooling circulation pump to cool down to 0°C and the water bath to heat up to 36°C before evaporation begins; Step 4, rinse the distillation flask with dichloromethane (5 ml × 3), transfer the lipids to a 20 ml vial, air dry naturally or blow dry with nitrogen, and further extract the neutral supernatant containing n-alkanes using n-hexane solution through silica gel column chromatography, and wait for air drying.

[0029] Test Case A total of 6 samples of Examples 1-5 and Comparative Example 1 were all fixed to 50 μL and placed in a gas chromatograph to determine the normal alkane content; Manual integration was performed based on the chromatogram obtained from the test, and the content of normal alkanes was calculated, and the results shown in Table 1 were obtained: Table 1 Extraction amount of normal alkanes at different digestion temperatures

[0030] Figure 1 The results showed that after microwave digestion, the content of extracted n-alkanes increased with the increase of microwave digestion temperature, and reached a maximum value when the digestion temperature reached 80°C. When the temperature was greater than 80°C, the content of n-alkanes decreased significantly. This may be due to the degradation of n-alkanes due to the high temperature. Therefore, the optimal digestion temperature for extracting n-alkanes from stalagmites proposed in the present invention is 80°C.

[0031] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for extracting normal alkanes from stalagmites, characterized in that: The following steps are involved: Step 1, taking a stalagmite sample, grinding it and performing acid hydrolysis to form a stalagmite digestion solution; Step 2, heating the stalagmite digestion solution to 40° C. to 80° C., keeping the temperature, performing a digestion reaction with microwave assistance, and cooling after the reaction to form a solution to be extracted; Step 3, mixing the liquid to be extracted with the extractant and performing multiple extractions, and concentrating to obtain a solid lipid extract; Step 4, separating the lipid extract by silica gel column chromatography, and extracting the normal alkanes in the stalagmite sample after air drying.

2. The method for extracting normal alkanes from stalagmites according to claim 1, characterized in that: In step 1, hydrochloric acid is used to acid hydrolyze the stalagmite sample, and the concentration of the hydrochloric acid is 2.5-3.5 mol / L.

3. The method for extracting normal alkanes from stalagmites according to claim 2, characterized in that: The mass volume ratio of the stalagmite sample and hydrochloric acid in step 1 is 3: (18~30) g / mL.

4. The method for extracting normal alkanes from stalagmites according to claim 1, characterized in that: The heating temperature in step 2 is 60°C to 80°C.

5. The method for extracting normal alkanes from stalagmites according to claim 4, characterized in that: In step 2, the time for heating to the insulation temperature is 3 to 7 minutes, and the insulation time is 15 to 25 minutes.

6. The method for extracting normal alkanes from stalagmites according to claim 1, characterized in that: After the microwave-assisted digestion reaction in step 2 is completed, the mixture is cooled to below 20° C.

7. The method for extracting normal alkanes from stalagmites according to claim 1, characterized in that: In step 3, the extractant is selected from dichloromethane, n-hexane, methanol or acetone.

8. The method for extracting normal alkanes from stalagmites according to claim 1 or 7, characterized in that: In step 3, the lipid extract is concentrated by evaporating the extractant.

9. The method for extracting normal alkanes from stalagmites according to claim 8, characterized in that: Step 3: Evaporate the extractant at 30-40°C until the extractant is completely volatilized.

10. The method for extracting normal alkanes from stalagmites according to claim 1, characterized in that: The eluent used for silica gel column chromatography separation in step 4 is n-hexane, cyclohexane or petroleum ether.

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