TITLE: Extraction of sediment samples using a modified Bligh and Dyer method: Hinrichs Lab
AUTHORS: Christopher CK Klaembt, Lennart Stock LS

[DESCRIPTION]
This updated Bligh and Dyer method is based on Xavier Prieto's protocol "OrgGeochem - Met007.1- Extraction of sediment samples using the Bligh and Dyer method" (review by Marcus Elvert, dated 01.01.2006). Since the original 2006 protocol, there have been several small changes and minor adjustments. This protocol outlines a way to perform the Bligh and Dyer extraction with modern equipment and provides useful tips and tricks.

The protocol includes the following steps:

Freeze-drying of sediment samples.
Performing the Bligh and Dyer extraction on sediment samples.
Evaporation and storage of the total lipid extract (TLE).

[BEFORE_START]
Useful tips and tricks: 
Pay attention to the glass stoppers, which may break due to overpressure in the separation funnel. It is better to use the aluminum foil from the combustion as lids during extraction, and only use the stoppers during liquid-liquid extraction.
For a longer phase separation, add DCM and MilliQ already after the first extraction step.
For 250 mL separation funnels, the best Teflon valves are the ones with the larger hole. Work best with the small O-rings.
Remember to clean the six-way gas tap with DCM:MeOH 5:1 and let it dry with minimal gas flow.
During drawing off of the organic layer in the separation funnel, it is better to leave a small amount of the organic layer instead of risking also drawing off any of the aqueous phases.
Distinctly mark and label the Teflon tube, separation funnel, Schott bottle, and TurboVap
The vial will be used for the same sample.

[GUIDELINES]
Freeze drying of sediment samples
Modified Bligh and Dyer extraction of sediment samples
Evaporation and storage of total lipid extract (TLE)

[STEPS]
SECTION: Freeze drying of sediments
0.38.       Take your samples out of the freezer
SECTION: Freeze drying of sediments
0.38.       Cover the container with aluminum foil 
SECTION: Freeze drying of sediments
0.38.       Poke little holes in the foil (syringe needles/ Pasteur pipettes work best)
SECTION: Freeze drying of sediments
0.38.       Follow freeze dryer instructions you find in the manual next to the machine
SECTION: Freeze drying of sediments
1. Freeze your samples (< -20°C) 
3. Cover the container with aluminium foil 

2. Take your samples out of the freezer

4. Poke little holes in the foil (syringe needles/ Pasteur pipettes work best)
SECTION: Bligh and Dyer procedure
6. Measure out X.XX grams of freeze-dried sediment into a 100 ml Teflon centrifuge vial
SECTION: Bligh and Dyer procedure
8. Add Bligh and Dyer phosphate extraction mix (5-10 mL per 1 g of freeze-dried sediment)
5. follow freeze dryer instructions you find in the manual next to the machine
SECTION: Bligh and Dyer procedure
7. Add the standards to the sediment 
SECTION: Bligh and Dyer procedure
7.1. For LC-MS analysis, add e.g. PC21 (0.5 µg / 1 g of freeze-dried sediment)
SECTION: Bligh and Dyer procedure
7.2. For GC-MS analysis, add GC-STD-Mix (1 µg / 1 g of freeze-dried sediment)
SECTION: Bligh and Dyer procedure
9. Sonicate for 10 minutes in an ultrasonic bath 
SECTION: Bligh and Dyer procedure
10. Centrifuge for 10 minutes at 2000rpm 
SECTION: Bligh and Dyer procedure
11. Decant the supernatant (solvent) into the separation funnel with closed valves!!! (use
Pasteur pipette if necessary) 
SECTION: Bligh and Dyer procedure
12. 
Repeat steps 3 to 6 one more time (1x) with the same amount of phosphate buffer

SECTION: Bligh and Dyer procedure
13. Repeat steps 3 to 6 two more times (2x) using the TCA buffer instead 
SECTION: Bligh and Dyer procedure
14. Add DCM and MilliQ to the separation funnel to separate the solution (5-10 mL DCM and 5-10 mL MilliQ (or sat. NaCl solution for faster separation) per 1 g of freeze-dried sediments)
SECTION: Bligh and Dyer procedure
15. Invert/shake the separation funnel to mix and wait for layers to separate – they can be cloudy, and it could take several minutes 
SECTION: Bligh and Dyer procedure
16. Draw off the organic layer (bottom) into a pre-combusted 250 mL Shott bottle
SECTION: Bligh and Dyer procedure
17. Extract the aqueous fraction three times (3x) with DCM (5-10 mL per 1 g freeze-dried
sediment) and combine DCM extracts with the  DCM layer (discard aqueous fraction)

SECTION: Bligh and Dyer procedure
18. Extract the organic fraction 3 times (3x) with MiliQ (5-10 mL per 1 g freeze-dried sediment) each time
discarding aqueous fraction – this step is not necessarily mandatory but helps clean the TLE

SECTION: Bligh and Dyer procedure
19. Transfer the TLE extract from the glass container to an 200 mL TurboVap vial (wash Shott bottle several times with DCM and add to TurboVap vial until clean)

SECTION: Bligh and Dyer procedure
20. Evaporate DCM from the total lipid extract under a flux of nitrogen in the
TurboVap evaporator to ~1 mL at no more than 37 °C 

SECTION: Transfer into 4 mL vial and drying over Sodium sulphate
21.  After evaporation, transfer the TLE into a new labeled and weighted (with lids) pre-combusted 4 mL vial. Again, wash the sample residues in the TurboVap vial with DCM until clean
SECTION: Transfer into 4 mL vial and drying over Sodium sulphate
22. Evaporate the TLE solution to complete dryness (unless you still observe water in your TLE to continue with 23.1) under the six-way gas tap under the fume hood. For faster evaporation, use the heating block and a heating plate at no more than 37°C
SECTION: Transfer into 4 mL vial and drying over Sodium sulphate
23. Following steps are also not necessarily mandatory, only if you feel or see that your TLE still contains water
SECTION: Transfer into 4 mL vial and drying over Sodium sulphate
23.1. Evaporate the TLE to ~0.5 mL under the six-way gas tap under the fume hood to complete dryness (remove as soon the sample is dry). For faster evaporation, use the heating block and a heating plate
SECTION: Transfer into 4 mL vial and drying over Sodium sulphate
23.2. Wash Pasteur pipette packed with sodium sulfate 3–4 times with DCM 
SECTION: Transfer into 4 mL vial and drying over Sodium sulphate
23.3. Put new labeled and weighted (with lids) pre-combusted 4 mL under the Pasteur pipette and pass TLE through the pipette to get rid of residual water (wash 4 mL vial with DCM until clean)
SECTION: Transfer into 4 mL vial and drying over Sodium sulphate
23.4. Pass as much DCM through the column as needed to fill the 4 mL vial

SECTION: Transfer into 4 mL vial and drying over Sodium sulphate
23.5. Evaporate the TLE under the six-way gas tap under the fume hood to complete dryness (remove as soon the sample is dry). For faster evaporation use the heating block and a heating plate
SECTION: Storage of TLE
24. Weigh the 4 mL vials with TLE and lids to calculate the yield

SECTION: Storage of TLE
25. Store dried TLE at -20 °C until analysis