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'.