TITLE: Phospholipid fatty acid (PLFA) analysis
AUTHORS: Eva Petrova, Roey Angel

[DESCRIPTION]
Phospholipid fatty acid (PLFA) purification and analysis

[BEFORE_START]
Supplies and Reagents
Extraction
            Supplies: Centrifuge bottles, Pipettes, test tubes, funnels, filter papers, Pasteur pipettes, beakers
            Reagents: phosphate buffer, chloroform, methanol, soil samples
Separation/Fractionation
            Supplies: vacuum, manifold, silica SPE columns 500 mg, test tubes, pipettes (including capillary pipettes), beakers
            Reagents: methanol, chloroform, acetone
Methylation
            Supplies: Vortex, water bath, test tubes, pipettes (0.5 mL, 2 mL, 1 mL, 10 mL), Pasteur pipettes, beakers
            Reagents: 0.2 N acetic acid, 0.2 N KOH in methanol, chloroform, toluene, chloroform-extracted water

[GUIDELINES]
NOTES:
All glassware must be fired in aluminum foil at 450°C for at least 4.5 hours to remove all organic substances.  Care must be used in organic-free technique to minimize contaminations between samples.  Any equipment which cannot be fired must be rinsed with methanol followed by chloroform and allowed to dry to remove any organics.  All transfers of solution MUST OCCUR IN A FUME HOOD.  Fatty acids are somewhat photosensitive, so care should be taken to minimize exposure to light.

[STEPS]
SECTION: Extraction of Lipids from Soils -First Extraction
1. Add 5 g of soil (fresh mass) to a centrifuge bottle (250 mL).
5 6
SECTION: Extraction of Lipids from Soils -First Extraction
2. Add 5 mL of chloroform, 10 mL of methanol, and 4 mL of phosphate buffer* minus the water content of the soil. 1 mL of chloroform for each g of soil, volumes should be 1:2:0.8 chloroform:methanol:buffer.
 
*50 mM phosphate buffer: 8.7 g K2HPO4 in 1 L D.I. water which has been chloroform extracted.  Adjust pH with 3.5 ml 6 N HCl to 7.4
5 2
10 2
4 2
SECTION: Extraction of Lipids from Soils -First Extraction
3. Swirl.  Extract wrapped in foil for 2 hours.
120 min
SECTION: Extraction of Lipids from Soils -First Extraction
4. Spin centrifuge bottles at 2500 rpm for 20-30 minutes.
 
20 min
SECTION: Extraction of Lipids from Soils - Second Extraction
5. Add additional 5 mL of chloroform and 5 mL of buffer to break the phase and achieve a final ratio of 1:1:0.9 chloroform : methanol : buffer.
5 2
5 2
SECTION: Extraction of Lipids from Soils - Second Extraction
6. Swirl.  Allow to separate overnight (approx 18 hours) covered to protect lipids from light.
1080 min
SECTION: Extraction of Lipids from Soils - Second Extraction
7. Spin centrifuge bottles at 2500 rpm for additional 20-30 minutes.
 
20 min
SECTION: Extraction of Lipids from Soils - Second Extraction
8. Transfer organic phase to a test tube through a Whatman #2 filter paper and evaporate solvent under N2.
SECTION: Extraction of Lipids from Soils - Second Extraction
9. Store in sealed test tubes under N2 at –20°C.
SECTION: Extraction of Lipids from Leaf Litter
10. Place 250 mg equivalent dry weight of ground litter in 20 mL test tube.
250 5
SECTION: Extraction of Lipids from Leaf Litter
11. Add 1.6 mL potassium phosphate buffer (subtract moisture in litter from the 1.6 mL of buffer), 4 mL methanol, 2 mL cholorform (solvents should be in final ratio of 0.8 : 2 : 1).
1.6 2
4 2
2 2
SECTION: Extraction of Lipids from Leaf Litter
12. Vortex each tube for 30 seconds.
30 s
SECTION: Extraction of Lipids from Leaf Litter
13. Place in 37°C water bath fro 30 minutes, vortexing every 5 minutes.
37 10
30 min
SECTION: Extraction of Lipids from Leaf Litter
14. Pipette out liquid (leave litter) into 2nd test tube.
SECTION: Extraction of Lipids from Leaf Litter
15. Add additional 1.6 mL buffer, 4 mL methanol, and 2 mL chloroform to original test tube. 
1.6 2
4 2
2 2
SECTION: Extraction of Lipids from Leaf Litter
16. Incubate at 37 °C for 30 minutes, vortexing every 5 minutes.
37 10
30 min
SECTION: Extraction of Lipids from Leaf Litter
17. Pipette out as much liquid as possible into 2nd test tube. Discard original test tube with leaf litter.
SECTION: Extraction of Lipids from Leaf Litter
18. Add additional 4 mL chloroform and 4 mL of buffer to second tube. Vortex for 30 seconds.
4 2
4 2
 
SECTION: Extraction of Lipids from Leaf Litter
19. Allow to separate overnight.  Remove lower phase only to small test tube by passing through a Whatman #2 filter paper.
SECTION: Extraction of Lipids from Leaf Litter
20. Store tubes, sealed, under N2 at –20 °C. 
SECTION: Fractionation of Lipids
21. Prepare columns by flushing them first with methanol to dehydrate them, then with chloroform to prepare them for the lipid.  Pull the solvents through with a very low vacuum*.
 
*Vacuum should be 5” Hg or less or the column may collapse.  The prep rinses and chloroform and acetone extractions may be collected in a waste container in the manifold and discarded before inserting test tubes for the methanol elution.
SECTION: Fractionation of Lipids
22. Allow lipids to reach room temperature. Resuspend the dried lipids in 150 μL of chloroform and transfer to the column.  Repeat for a total of 4 transfers per sample. 
150 1
SECTION: Fractionation of Lipids
23. Elute neutral lipids with 5 mL of chloroform. Pull solvent through with a low vacuum.  Discard solvent. 
5 2
SECTION: Fractionation of Lipids
24. Elute glycolipids with 5 mL of acetone. Pull solvent through with a low vacuum.  Discard solvent. 
5 2
SECTION: Fractionation of Lipids
25. Elute phospholipids with 5 mL of methanol under vacuum into a test tube. 
5 2
SECTION: Fractionation of Lipids
26. Blow off solvent (methanol) with the N2 Store dried PLFAs under N2 at –20 °C until methylation.
SECTION: Methylation- formation of fatty acid methyl esters
27. Remove samples from freezer and allow them to reach room temperature.
SECTION: Methylation- formation of fatty acid methyl esters
28. Make 0.2 N methanolic KOH by dissolving 0.28 g KOH in 25 mL of methanol. KOH absorbs water from the air, so movement from the balance to the methanol is critical.  Make fresh every day. 
25 2
SECTION: Methylation- formation of fatty acid methyl esters
29. Make 0.2 N acetic acid if none already made by diluting 1.15 mL glacial acetic acid to 100 mL total volume with chloroform extracted D.I.
100 2
SECTION: Methylation- formation of fatty acid methyl esters
30. Create 1:1 v mix of methanol : toluene.
SECTION: Methylation- formation of fatty acid methyl esters
31. Add 0.5 mL of the methanol : toluene solution to each sample.
0.5 2
SECTION: Methylation- formation of fatty acid methyl esters
32. Add 0.5 mL of KOH in methanol to each sample. Vortex for 30 seconds.
0.5 2
 
SECTION: Methylation- formation of fatty acid methyl esters
33. Place in water bath heated to 37 °C for 15 minutes. Then cool tubes to room temperature.
37 10
15 min
SECTION: Methylation- formation of fatty acid methyl esters
34. Add 0.5 mL of acetic acid to each tube.   May look milky/chunky. 
0.5 2
SECTION: Methylation- formation of fatty acid methyl esters
35. Add 2 mL of chloroform to all samples followed immediately by 2 mL of chloroform-extracted DI water. Vortex for 30 seconds.
2 2
2 2
 
SECTION: Methylation- formation of fatty acid methyl esters
36. Centrifuge samples for 5 minutes.  
 
SECTION: Methylation- formation of fatty acid methyl esters
37. Pipette out bottom (organic) layer into a new test tube being careful not to transfer any of the aqueous phase.
SECTION: Methylation- formation of fatty acid methyl esters
38. Add 1 mL of chloroform to each of the original sample tubes.  Vortex for 30 seconds.  Centrifuge for 5 minutes.  Pipette organic (lower) layer into the new test tube. 
1 2
 
 
SECTION: Methylation- formation of fatty acid methyl esters
39. Repeat step 38.
   
SECTION: Methylation- formation of fatty acid methyl esters
40. Add 1 mL of chloroform to each of the original test tubes but DO NOT VORTEX OR CENTRIFUGE.  Transfer chloroform to new test tubes.  New test tubes should now contain FAMEs in 5 mL of chloroform Remove chloroform under N2 blow-down.  Store samples at –20 °C until identified by gas chromatography.
1 2
SECTION: Separation, Quantification and Identification
41. Resuspend FAMEs in hexane. Include a known amount of an internal standard.
SECTION: Separation, Quantification and Identification
42. Transfer to GC vial with PTFE septa cap.
SECTION: Separation, Quantification and Identification
43. Identify by retention time/co-elution with standards on a GC 
SECTION: Separation, Quantification and Identification
44. Molar concentrations of each fatty acid can be determined by relationship between area of standard peak and known concentration of standard.