Lipid extractions and chemical determinations

Protocol

Authors

Hendrik Poorter, Yvonne de Jong-Van Berkel

Author Affiliations

Hendrik Poorter IBG-2, Forschungszentrum Jülich

Yvonne de Jong-Van Berkel, Ecophysiology of Plants, Faculty of Science, Utrecht University

Overview

This protocol follows the Bligh and Dyer (1959) method.

Lipids occur in the membranes of plant material. There are wide ranges of lipids which can be separated on their specific properties. With this method we are only interested in the total amount of lipids in plant material. Lipids are apolar compounds, which can be separated in apolar solvents such as chloroform or ether. After extraction, the lipids can be measured gravimetrically.

Extraction

Materials/Equipment

Reagents

  • 100% Methanol
  • Chloroform
  • Deionized water

Equipment

  • Labtop centrifuge
  • Centrifuge tubes (15 ml)
  • Heating block
  • Nitrogen gas cylinder
  • Stove

Procedure

  1. Weigh 50 mg dry, ground plant material or 500 mg fresh plant material in a centrifuge tube.
  2. Add deionized water, 1 ml to dry plant material and fill up to 1 ml in case of fresh plant material (500 mg fresh weight gives about 0.45 ml water, so 0.55 ml deionized water must be added).
  3. Add 2 ml methanol and 1 ml chloroform.
  4. Mix thoroughly on a vortex.
  5. Add 1 ml deionized water and 1 ml chloroform.
  6. Mix again on a vortex.
  7. Centrifuge for 10 minutes at 4500 rpm (2649 g).
  8. Now there is a two-phase extract, water/methanol above, chloroform below, separated by a dense protein layer.
  9. Remove the chloroform (lowest layer) with a Pasteur pipette.
  10. Extract the water/methanol two more times with 1 ml chloroform.
  11. Remove the chloroform after centrifugation at 4500 rpm (2649 g).
  12. The combined chloroform is the extract containing the lipids that can be determined gravimetrically after evaporation of the chloroform.

Chemical Determination

Materials/Equipment

  • Benchtop centrifuge
  • Centrifuge tubes
  • Heating block with nitrogen gas cylinder
  • Stove

Procedure

Determination of total lipids in chloroform extract

  1. Evaporate the chloroform of the extract.
  2. To do so, place the centrifuge tubes in a heating block of 40∘C. A nitrogen gas flow in the tubes decreases the evaporating time (a simple construction of tubes and Pasteur pipettes is sufficient).
  3. After removal of the chloroform the lipid fraction must be dried in a stove at 70∘C for 24 hours.
  4. Lipids are determined gravimetrically by weighing the centrifuge tubes with the lipid fraction.

Calculations


- File (1)

where

Wc = weight empty tube

Wc+l = weight tube with lipids

Wsample = weight sample

Notes and troubleshooting tips

  • The Bligh and Dyer method is especially designed for ‘wet’ material. In the case of dry material it may be good to first presoak the plant material in some water.
  • Apart from lipids, this method will also include lipopolysaccherides and other compounds.

Reference values

In leaf material, 20 – 70 mg g-1, lower in stems and roots. Fruit sometimes can contain considerably more lipids, up to 450 mg g-1 (Poorter & Villar 1997).

Literature references

Bligh, E.G. & Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Phys. 37: 911-917.

Osborne, D.R. & Voogt, P., 1978. The analysis of nutrients in foods. Academic Press, London. pp: 161-163.

Poorter, H & Villar, R. 1997. The fate of acquired carbon in plants: chemical composition and construction costs. In: Plant Resource Allocation. Academic Press, pp 30-72.

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