3.10: Sonication Extraction of Lipid Biomarkers from Sediment

1. Collect the Necessary Materials

1. Samples (leaves, dirt, fungi, bark, tissue), usually frozen, freeze-dried, crushed, and homogenized prior to extraction, extracted in groups to maximize efficiency. Extract three samples.
2. Depending on the size of the sample, vials with volumes ranging from 4-60 mL may be used. For this experiment, borosilicate glass vials (40 mL) and solvent safe caps are used. Vials, borosilicate glass pipettes, and weighing tins should be combusted at 550 °C for 6 h prior to ensure removal of possible organic contaminants.
3. Dichloromethane and methanol are common in most organic geochemistry laboratories. They are used individually to rinse lab tools and glassware before use. A mixture of dichloromethane (DCM) to methanol (MeOH; 9:1) is used in many labs to efficiently extract biomarkers with a wide range of polarities. Solvents should be free of organic contaminants.
4. Use a sonication water bath at room temperature. A range of sonicator sizes and sonication with or without heat are available from major scientific equipment retailers.
5. Vial racks, which should be waterproof, will be placed in sonication bath.
6. Solvent approved chemical hood.

2. Preparation of Sample

1. Place a combusted weighing tin on the lab scale and then tare.
2. Rinse the lab spatula with solvent, then use it to transfer an appropriate mass of sample into the weighing tin, and record the mass.
   1. The mass of the sample varies depending on its organic matter content. Relatively organic matter lean material (marine mud) may require several grams, while organic matter rich material (leaf tissue) may require much less.
3. Transfer all of the material in the weighing tin into a combusted, pre-weighed, and labelled vial. Cap the vial, then discard the weighing tin.
4. Perform steps 2.1 - 2.3 for each sample to be extracted.

3. Extraction

1. Using a pre-combusted pipette and bulb, transfer ~20 mL of the DCM:MeOH (9:1) mixture into each vial (vial should be approximately half full). Recap vials before moving on to next sample, so the volatile solvents do not evaporate.
   1. The volatilities of DCM and MeOH are different. Evaporation of extraction solvent due to uncapped sample vials has the ability to change its polarity, and thus the extraction.
2. Place the sample vials into a waterproof vial rack.
3. Check that the level of water in the sonication bath is only deep enough to submerge the sample vials up to the top of the extraction solvent. Too much water may cause the vials to float; too little water may stop the samples from being properly agitated.
4. Place the vial rack with the samples in it directly into the sonication bath.
5. Sonicate for 30 min at room temperature.
6. Remove sample rack from sonicator. If extracting sediments, let it set for 30 min to allow settling to occur. If extracting another set of samples, put in sonicator at this time.
7. Remove the DCM:MeOH mixture from the extraction vial, using a pre-combusted pipette and bulb, and insert into another pre-weighed, pre-combusted, and labeled 40-mL vial.
8. Repeat 3.1 - 3.7 3x for all samples.
9. Allow extracted samples to dry in their vials, caps off, and in the hood, covered loosely with a piece of foil. Label as 'extracted residue' and store.
10. Label the combined extracts for each sample as 'TLE'.

The first step in paleoclimatology is to collect, or extract, the biomarkers from the sediment they are found in. Environmental samples are composed of non-organic components, such as minerals, water, and metals, and organic components that are created by living organisms in the area. Before these organic components can be used by scientists to elucidate information about the past, they must be removed from their environment. Sonication, which utilizes ultrasonic waves, is the simplest and least expensive of these techniques.

This video is part of a series on lipid extraction, purification, and analysis from sediments. It will illustrate lipid extraction by ultrasound and present a few applications of the method.

Because of the wide range of biomarkers, there is no single solvent optimized to extract all of them. This is summarized by the so-called 'like dissolves like' rule, whereby relatively apolar molecules dissolve in apolar solvents such as dichloromethane, and more polar molecules dissolve in more polar solvents such as methanol. Solvent mixtures for the extraction of specific lipids or groups of lipids are generally optimized empirically.

To accelerate extraction and increase yield, a sonication system is used to apply ultrasound - waves with frequencies greater than 20 kHz, in conjunction with the solvent mixture. When these waves contact the liquid organic phase, they cause the formation of short-lived microbubbles of solvent vapors that rapidly grow and collapse. On collapsing, these bubbles release a tremendous amount of energy as mechanical shear, facilitating lipid solubilization and dramatically increasing the efficiency of extraction.

After the ultrasound assisted solvent-extraction process, the result is a crude extract preparation, called a total lipid extract, that is subjected to further purification to allow qualitative and quantitative examination of lipid signatures. Now that you understand some of the main principles behind lipid extraction by sonication, lets take a look at a protocol for how the procedure is performed.

Collect the necessary sample materials from a chosen location. Some examples are lacustrine and marine sediments, terrestrial soils, microbial cultures, or plant leaves. Collected material is frozen overnight. Following this it is freeze-dried in a freeze dryer for 2 to 3 days. Crush and homogenize the freeze-dried samples prior to extraction with a solvent-rinsed mortar and pestle. To remove organic contaminants, combust the required borosilicate glass pipettes, vials, and weighing tins in an oven. After allowing the glassware to cool in the oven, rinse the metal tools with a mixture of dichloromethane and methanol. Once the sample and glassware are prepared, the sonication procedure can begin.

From this step on, all containers and glassware should be combusted before use. Place the weighing tin on a scale and tare. Rinse the lab spatula with the solvent mixture, then, use it to transfer an appropriate mass of freeze-dried, homogenized sample into the weighing tin and record the mass. Carefully transfer the weighed sample into a labeled vial. Using the squirt bottle of DCM:MeOH, add enough that the sample is covered by 1 to 2 cm of solvent, and cap the vial. Place the vial on a waterproof rack, now ready for sonication. Place the rack directly into the sonication bath. Check that the water level in the sonication bath is just deep enough to submerge the sample vials up to the top of the extraction solvent. Sonicate for 30 minutes at room temperature. After sonication, remove the rack from the sonicator. Let the vials sit to allow sediment settling to occur.

Remove the dichloromethane-methanol upper phase from the extraction vial using a pipette and bulb, and transfer into another pre-weighed and labeled vial. Repeat the sonication process a total of three times for each sample. Collect the extracts into one vial. Allow extracted samples to dry in their vials, caps off, and in the hood, covered loosely with a piece of foil. Label as 'extracted residue' and store in the extraction solvent. Now that the biomarkers have been extracted, they must be purified before analysis can take place.

Sonication accelerates several solvent extraction processes and is widely used in geochemical studies. Many archeologists work with geochemists in order to reconstruct the environmental and cultural circumstances under which early human civilizations lived. Pottery, one of the oldest human inventions, when unearthed, can be found to contain residual molecular fossils from wine, rice, or other contents that were once stored within.

To unearth chemical evidence of substances absorbed onto the surfaces, small samples of pottery are sonicated in the presence of organic solvents and extracted compounds can be subsequently identified downstream by spectroscopic methods. This kind of analysis helps archeologists detect the kinds of resources that were available to ancient populations and reconstruct the conditions of their habitat.

Photosynthetic microalgae are found in marine and freshwater ecosystems. Because they grow in seawater-based media and their culture occupies significantly smaller areas, they are now being widely studied as a promising alternative to terrestrial plants for the production of biofuels.

To extract lipids from a microalgal biomass, these researchers describe a sonication-assisted solvent extraction. Acoustic cavitation during sonication effectively disrupts rigid microalgal cell walls in order to liberate lipids. Such techniques aid the characterization of new microalgae from the environment for the production of non-petroleum sources of energy.

You've just watched JoVE's introduction to Sonication-Assisted Extraction of Biomarkers from Sediments. The following videos will explain how the extract is further purified for analysis.

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