Sep 16, 2024
Phospholipid fatty acid (PLFA) analysis
- 1Soil and Water Research Infrastructure
- Anaerobic and Molecular Microbiology Lab, Biology Centre CASTech. support email: eva.petrova@bc.cas.cz
Protocol Citation: Eva Petrova, Roey Angel 2024. Phospholipid fatty acid (PLFA) analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrj7ogmkn/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 24, 2018
Last Modified: September 16, 2024
Protocol Integer ID: 12410
Abstract
Phospholipid fatty acid (PLFA) purification and analysis
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.
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
Extraction of Lipids from Soils -First Extraction
Extraction of Lipids from Soils -First Extraction
Add 5 g of soil (fresh mass) to a centrifuge bottle (250 mL).
5 g soil (fresh mass)
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 mL chloroform
10 mL methanol
4 mL phosphate buffer (50mM, pH 7.4)
Swirl. Extract wrapped in foil for 2 hours.
02:00:00 extraction
Spin centrifuge bottles at 2500 rpm for 20-30 minutes.
Command
2,500 rpm
00:20:00
Extraction of Lipids from Soils - Second Extraction
Extraction of Lipids from Soils - Second Extraction
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 mL chloroform
5 mL phosphate buffer
Swirl. Allow to separate overnight (approx 18 hours) covered to protect lipids from light.
18:00:00 extraction in dark
Spin centrifuge bottles at 2500 rpm for additional 20-30 minutes.
Command
2,500 rpm
00:20:00
Transfer organic phase to a test tube through a Whatman #2 filter paper and evaporate solvent under N2.
Store in sealed test tubes under N2 at –20°C.
Extraction of Lipids from Leaf Litter
Extraction of Lipids from Leaf Litter
Place 250 mg equivalent dry weight of ground litter in 20 mL test tube.
250 mg dry weight of ground litter
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 mL potassium phosphate buffer
4 mL methanol
2 mL chloroform
Vortex each tube for 30 seconds.
00:00:30 vortex
Place in 37°C water bath fro 30 minutes, vortexing every 5 minutes.
37 °C water bath
00:30:00 vortexing every 5 minutes
Pipette out liquid (leave litter) into 2nd test tube.
Add additional 1.6 mL buffer, 4 mL methanol, and 2 mL chloroform to original test tube.
1.6 mL phosphate buffer
4 mL methanol
2 mL chloroform
Incubate at 37 °C for 30 minutes, vortexing every 5 minutes.
37 °C water bath
00:30:00
Pipette out as much liquid as possible into 2nd test tube. Discard original test tube with leaf litter.
Add additional 4 mL chloroform and 4 mL of buffer to second tube. Vortex for 30 seconds.
4 mL chloroform
4 mL phosphate buffer
Command
30s
Allow to separate overnight. Remove lower phase only to small test tube by passing through a Whatman #2 filter paper.
Store tubes, sealed, under N2 at –20 °C.
Fractionation of Lipids
Fractionation of Lipids
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.
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 µL chloroform
Elute neutral lipids with 5 mL of chloroform. Pull solvent through with a low vacuum. Discard solvent.
5 mL chloroform
Elute glycolipids with 5 mL of acetone. Pull solvent through with a low vacuum. Discard solvent.
5 mL acetone
Elute phospholipids with 5 mL of methanol under vacuum into a test tube.
5 mL methanol
Blow off solvent (methanol) with the N2 Store dried PLFAs under N2 at –20 °C until methylation.
Methylation- formation of fatty acid methyl esters
Methylation- formation of fatty acid methyl esters
Remove samples from freezer and allow them to reach room temperature.
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 mL 0.2N methanolic KOH
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 mL 0.2 N acetic acid
Create 1:1 v mix of methanol : toluene.
Add 0.5 mL of the methanol : toluene solution to each sample.
0.5 mL methanol : toluene solution
Add 0.5 mL of KOH in methanol to each sample. Vortex for 30 seconds.
0.5 mL KOH in methanol
Command
30s
Place in water bath heated to 37 °C for 15 minutes. Then cool tubes to room temperature.
37 °C water bath
00:15:00
Add 0.5 mL of acetic acid to each tube. May look milky/chunky.
0.5 mL acetic acid
Add 2 mL of chloroform to all samples followed immediately by 2 mL of chloroform-extracted DI water. Vortex for 30 seconds.
2 mL chloroform
2 mL chloroform-extracted DI water
Command
30s
Centrifuge samples for 5 minutes.
Command
5 minutes
Pipette out bottom (organic) layer into a new test tube being careful not to transfer any of the aqueous phase.
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 mL chloroform
Command
30s
Command
5 min
Repeat step 38.
go to step #38
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 mL chloroform
Separation, Quantification and Identification
Separation, Quantification and Identification
Resuspend FAMEs in hexane. Include a known amount of an internal standard.
Transfer to GC vial with PTFE septa cap.
Identify by retention time/co-elution with standards on a GC
Molar concentrations of each fatty acid can be determined by relationship between area of standard peak and known concentration of standard.