Standard Operating Procedure
Lipid extraction procedure
Department: Agronomy
Created by: Vidya Iyer
Laboratory: Crop Production & Physiology
Supervisor: Dr. Mark Westgate
Lab Supervisor: Maria Hartt Eckerman me
Date approved:
11 July 2005
Procedure Overview: Lipid extraction of neutral lipids carried out using hexane.
Equipment and reagents necessary:
Borosilicate glass tubes, 5 per sample
when air drying or using hot water bath, use 13x75 mm tubes
when drying with heat blocks, use 10x75 mm tubes
Tube rack(s)
Weigh balance
Heating block/ Water bath
Freeze-dried ground soybean embryo tissue
Microcentrifuge tubes
Mortar and pestle
Pipette and tips, 1 ml
Hexane (HPLC grade, Fisher Scientific H-302)
Benchtop microcentrifuge
Fume hood
Vortex
-20C freezer
Protocol:
Sample weight should be in the range of 50 to 100 mg when using microcentrifuge
tubes and the heating block unit. Use freeze-dried, ground embryo tissue (seed coat
removed prior to freeze-drying). Embryo can be ground using a mortar and pestle.
1. Label and pre-weigh disposable, borosilicate glass tubes (5 times number of
samples for 5 sets of hexane washes)

when air drying or using hot water bath, use 13x75 mm tubes

when drying with heat blocks, use 10x75 mm tubes
2. Preheat heating block to 40 0C.
3. Weigh freeze-dried ground soybean embryo tissue in properly labeled
preweighed microcentrifuge tube
4. Using a pipette, add 1 ml high grade hexane to each microcentrifuge tube and
vortex
5. Place tubes in the heating block at 40C for about an hour
6. Centrifuge 1 min in benchtop microcentrifuge at high speed
7. Remove supernatant (leaving a small amount in the microcentrifuge tube) to
preweighed labeled glass tubes; keep tube rack under hood
8. Repeat steps 4-7 4 times, placing supernatant in new sets of preweighed tubes
each time for a total of 5 tubes per sample
9. Place microcentrifuge tube containing resulting pellets in heating block (turned off
but cooling over night).
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10. Store defatted pellet for further protein/starch analyses at -20C freezer in 1522
Agronomy in properly labeled, covered container.
11. Weigh glass tubes of supernatant when they have come to room temperature
and dryness (about 48 hours). Subtract the pre-weight and sum the differences
to determine the total weight of oil extracted.
12. Determine percent of original sample weight. Correct to 13% moisture content.
Assume freeze-dried tissue is 0% moisture content
Formulae for calculations:

% moisture content correction:
(1-moisture content initial ) x tissue weight initial = tissue weight final
(1-moisture content final)

% oil calculation:
mg oil
x 100 = % oil final
(tissue weight final) mg
Note: 5 separate sets of tubes for collecting the supernatant from each extraction is
mainly to estimate the extraction efficiency. Once familiar with the procedure, one set
of tubes is enough and the supernatant from all the five washes can be collected in a
single tube for each sample.
Personal Protective Equipment / Engineering Controls:
Eye protection (goggles & shield)
Skin protection (proper shoes, nitrile gloves, lab coat, etc.)
Ventilation system
Safety shower
Eye wash station
Hazard Controls & Storage Precautions:
Hexane: Always wear safety glasses. Do not work in an area in which sources of
ignition, such as a Bunsen burner or hot air gun, are used. Ensure good
ventilation at all times - use a fume hood for your work if possible
Waste Disposal Procedures & Decontamination:
Hexane: Does not dissolve in water and must not be flushed down the drains. Store
in a suitable container (located in a well-ventilated area) for later disposal. Do not
use sawdust or other combustible materials. Keep any ignition sources away In
case of a spill.
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Health & Safety Info for Required Reagents:
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Incompatibilities
Keep away from
heat, flame and
sources of ignition.
Keep from contact
with oxidizing
materials.
The above summary consists of guidelines for proper handling & disposal
of chemicals used in this procedure. You must read attached MSDSs for
more specific information before using the procedure.
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