Organic Extraction

This protocol is used to prepare samples for GC-MS when analyzing organic compound degradation assays. The supernatant of bacterial cultures used in the assay is isolated and acidified to pH < 2 prior to extraction.

SAFETY GLASSES AND GLOVES MUST BE WORN!!!

1. Rinse all clean glassware (including separatory funnels, round-bottom flasks, and glass filter funnels)with acetone, and let air dry in the fume hood.
2. Fold a piece of filter paper in four, place in a funnel, and add enough anhydrous sodium sulfate to fill the folded filter paper about 3/4 to the top.
3. Add 10 mL n-hexadecane as the extraction standard to the acidified culture sample.
4. Add approximately 15 mL ethyl acetate to the acidified sample, and shake; pour into separatory funnel. Allow the aqueous and solvent layer to separate. Ethyl acetate will be in the top layer.
5. Drain the bottom aqueous layer back into the sample vessel, taking care to not drain the ethyl acetate layer by closing the stopcock when the aqueous layer has drained out.
6. Drain the ethyl acetate layer through the filter paper containing sodium sulfate, and allow it to collect into the round-bottom flask.
7. Repeat the extraction procedure 2 more times, for a total of 3 times. Rinse the sodium sulfate with several pipets-full of fresh ethyl acetate.
8. Concentrate the collected ethyl acetate layers by rotary evaporation to a volume about the size of a quarter (1-2 mL).
9. Quantitatively transfer the concentrated layer into an autosampler vial for analysis.
10. Wash all glassware with soap and water using a brush, and rinse many times with regular tap water and distilled water and allow to air dry on drying rack.
11. Analyze samples by GC-MS (gas chromatography-mass spectrometry) and compare the carbazole peak to the hexadecane peak area ratios to determine the extent of the biodegradation of carbazole.