Introduction

There are many different methods for extracting organic compounds. This recipe outlines the one that has become standard in our lab, using the microwave-accelerated solvent extraction system (MARS-X). It is a very general method that works well for extracting hydrocarbons and and lipids of moderate polarity (including fatty acids) from sediments and rocks. Many modifications are possible to optimize the method for specific types of analytes. Typically, multiple samples (up to 10 or more) can be processed in parallel. An accompanying blank is recommended.

Materials

• 1-4g of solid sample
• teflon MARS vessel, cap, and plug
• teflon-coated stir bar
• 20mL of 9:1 dichloromethane/methanol
• silica gel (for a blank)

Warnings

• The MARS system heats organic solvents to temperatures well above their boiling point. Removing a sealed vessel from the system and opening it while at elevated temperature would be extremely hazardous to your health. Be certain that all vessels are cool before opening them!
• Even when the vessels are cool, a modest amount of solvent vapor is expelled when they are first opened. Be sure to handle the sample vessels in a fume hood, pointing the tops toward the back of the hood when they are first opened.

Procedure

1. Wet sediments should be dried before extraction. A drying oven set at ~60C will work, but may also promote microbial growth. Freeze drying is preferred. A method blank (~2g of precombusted silica gel or quartz sand) should accompany your samples throughout this entire procedure.
2. Samples should be ground to approximately fine-sand size. Sediments can be ground in a mortar and pestle, while rocks will require a jaw crusher, shatterbox, or other device. Be sure to clean all crushing hardware thoroughly with DCM or acetone before use and between samples. Note that we do not recommend the MARS extraction method for biological materials - a simple Bligh and Dyer type extraction in a separatory funnel is usually adequate for those.
3. Clean a teflon vessel, cap, plug, and stirbar by rinsing twice with DCM and wiping off any visible contamination with a Kim-wipe.
4. Weigh 1-4 grams of dry, crushed sample into the MARS vessel. Use more sample for organic-lean materials, less sample for organic-rich ones. If you want to extract large amounts of sample, distribute it between several vessels. All vessels should containg the same amount of sample (note A).
5. Add the precleaned stirbar to the sample, followed by 20mL of 9:1 dichloromethane/methanol (note B). Place the teflon plug into the top, then screw on the vessel cap. The cap should be hand-tightened with moderate strength, but do not overtighten! It is possible to deform the teflon and ruin the threads. All vessels must contain the same amount and type of solvent (note A).
6. Arrange the teflon vessels in the inner ring of the MARS carousel. Space them out as evenly as possible around the ring. A minimum of 6 vessels must be present for proper temperature regulation, so if necessary make up several "dummy" vessels by filling them with an equivalent amount of sand + solvent (note A).
7. Run the "normal" method on the MARS by choosing "load method", then "normal", then start. This method will heat the samples to 100C for 15 minutes with stirring.
8. After the method has finished, allow the vessels to cool for 20-30 minutes inside the carousel (note C). You may then remove them from the carousel and place them on the bench, or in an ice bath to cool them further. Wait until they are at room temperature before loosening the caps in a fume hood (see warnings above).
9. Filter the solvent + samples using extra 9:1 solvent to help transfer the solid material out of the sample vessel. Allow the vessel to dry in the fume hood before moving it to the sink area.
10. Clean the empty vessel, plug, cap and stir-bar in warm soapy water (note D), then rinse with distilled water. Air-dry, then return them to the appropriate drawer.

Notes

1. The MARS system monitors the temperature of the vessels using an infrared detector that looks at the bottom of the vessels in the carousel. The sensor averages the temperature of all the vessels, and the microwave power is adjusted to keep the average temperature at the setpoint. If there are too few vessels (<6), then the long gaps between temperature measurements cause problems with uneven heating. The heating rate of each individual vessel will depend on the amount and type of sample + solvent in the vessel. Most of the heating is due to absorption of microwave energy by the polar solvent, and for this reason all of the vessels in the carousel at one time must contain the same amount and type of solvent. Using different solvent mixtures, even if the volumes are identical, will lead to very different temperatures between vessels. Solid sample materials typically do not absorb microwave energy very strongly, so it is less critical (though still important) to try to match sample characteristics as closely as possible.
2. This solvent mixture is a good compromise that is suitable for extracting the majority of lipids from sediments. It is possible to substitute other solvent mixtures. For example, 4:1 hexane/acetone is a more nonpolar mixture that is suitable for hydrocarbons, while 1:2:1 DCM/methanol/water is more suitable for extracting polar lipids. The MARS application guide contains more information on picking solvent mixtures.
3. The walls of the teflon vessels are relatively weak, and require the woven kevlar sleeves to support them at high temperature. If you removed a vessel from its kevlar sleeve while at 100C, it is possible that the side walls would deform and ruin the vessel. Wait until the temperature sensor is reading 50C or lower before removing the vessels. It is then possible to cool them rapidly by placing them in a wash tub filled with ice.
4. It is important to prevent the teflon sample vessels from becoming scratched. Any scratches will lead to localized heating of the sample and eventually premature failure of the vessel. Usually just shaking the capped vessel with soapy water is enough to dislodge particles. If not, try using a paper towel or long-handled cotton swab. Do not use a bottle brush or test-tube brush, as these will scratch the teflon.