8. Crude Fat (Ether Extract) in Forages
Fat (Crude) or Ether Extract in Animal Feed. (920.29) Official
Methods of Analysis. 1990. Association of Official Analytical
Chemists. 15th Edition.
This method is applicable for the determination of crude fat in
dried forages and mixed feeds. It is not applicable for oilseeds,
baked and/or expanded products (pet foods), liquid feeds, sugar
products, and feeds containing dairy products. For determining
fat in oilseeds, consult Official Methods and Recommended
Practices of the American Oil Chemists Society.
A dried, ground sample is extracted with diethyl ether which
dissolves fats, oils, pigments and other fat soluble substances.
The ether is then evaporated from the fat solution. The resulting
residue is weighed and referred to as ether extract or crude fat.
Both the ether and the samples must be free of moisture to avoid
coextraction of water-soluble components in the sample such as
carbohydrates, urea, lactic acid, glycerol, etc. If water-soluble
components are present in large amounts in the sample, they are
washed out of the sample prior to drying. Low temperatures are
used to evaporate the ether and remove residual moisture to
prevent oxidation of the fat. Petroleum ether does not dissolve
all of the plant lipid material, and therefore it cannot be
substituted for diethyl ether.
Goldfisch fat extraction apparatus, 6-flask unit, equipped with
glass thimble holders and ether reclaiming tubes
Extraction thimbles, 22 x 80 mm, Alundum (porous clay), coarse
Fat beakers, pyrex, with ground lip, engraved with a number, 50 x
85 mm Drying oven, 102oC gravity convection Analytical balance,
sensitive to 0.1 mg Desiccator and tongs
Filter paper, Whatman #1, 11 cm, or equivalent
Steambath in a hood (optional) Gloves, white nylon, lintless
Anhydrous Diethyl Ether, purified for fat extraction Mallinkrodt
#0844 or equivalent. To prevent ether from absorbing water,
purchase it in small containers and keep containers tightly
- Ether has an extremely low flash point. Have no open
flames nearby. Avoid inhaling ether vapors. Store ether
in metal containers. Handle open containers (reagent cans
and fat beakers) in a hood. Conduct the extractions in a
well ventilated area.
- Peroxides can accumulate in open containers of ether.
These are explosive and shock sensitive. Check each
container opened for more than 30 days for peroxides.
Ether-containing peroxides must be disposed with special
- Electrical equipment is to be grounded. Extractors should
- Make sure all ether is evaporated from the beakers before
placing them in the oven to avoid a fire or explosion.
Procedure: Sample drying
- Weigh 1.5 to 2 g of ground sample into a thimble
recording the weight to nearest 0.1 mg (W1). Weigh a
second subsample for dry matter determination.
- Or -
1A) If the sample contains large amounts of
carbohydrates, urea, glycerol, lactic acid or
water-soluble components, weigh 2 g sample to nearest 0.1
mg (W1) into a small filter cone. Extract with five 20 mL
portions of deionized water allowing each portion to
drain, then insert the paper and sample into thimble.
- Dry for 5 hr at 100oC.
- Dry beakers to be used for fat determination for at least
1 hr at 100oC. Cool the appropriate number of fat beakers
in a desiccator. Weigh and record the weight to the
nearest 0.1 mg (W2).
- When the drying period is over, remove the samples from
the oven to a desiccator. (This is a convenient stopping
point. The samples should be stored in a desiccator if
not immediately extracted.)
- Line the fat beakers up in front of the extractor and
match the thimbles with their corresponding fat beakers.
- Slip the thimble into a thimble holder and clip the
holder into position on the extractor.
- Add about 40 mL of diethyl ether (one glass reclaiming
tube full) to each fat beaker.
- Wearing white gloves, slip the beaker into the ring clamp
and tightly clamp the beaker onto the extractor. If the
clamp is too loose, insert another gasket inside the
- Raise the heaters into position. Leave about a 1/4 inch
gap between the beaker and the heating element.
- Turn on the heater switch, the main power switch, and the
- After the ether has begun to boil, check for ether
leakage. This can be detected by sniffing around the ring
clamp. If there is leakage, check the tightness of the
clamp and if necessary replace the gasket(s).
- Extract for minimum of 4 hr on a Hi setting (condensation
rate of 5 to 6 drops per second), or for 16 hr on a Low
setting (condensation rate of 2 to 3 drops per sec).
- After extraction, lower the heaters, shut off the power
and water, and allow the ether to drain out of the
thimbles (about 30 min). This is a good stopping point.
Ether Distillation and Weighing of Fat Residue
- Remove the thimble from the holder, and rinse the holder
with a small portion of diethyl ether from the
washbottle. Clip an ether reclaiming tube in place and
reattach the fat beaker.
- Reposition the heaters and turn on the electricity and
water. Proceed to distill the ether using a Hi setting.
- Distill until a thin layer of ether remains in the bottom
of the beaker, and then lower the heater. Do not allow
beakers to boil dry. Overheating will oxidize the fat.
When the last beaker has finished, shut off the power and
- Wipe the exterior of the beaker clean with a Kimwipe as
it is being removed from the extractor.
- Empty the reclaiming tubes into the "USED"
diethyl ether container.
- Place the tray of beakers in an operating hood to finish
evaporating the ether. If there is no hurry, air moving
through the hood will be sufficient without heat. A
steambath may be used to speed up the evaporation.
Beakers should remain in the hood until all traces of
ether are gone. Carefully sniff each beaker to determine
if any ether remains.
- Place the beakers in a 102oC gravity convection oven.
Warning: If a beaker containing ether is placed in the
oven an explosion may occur.
- Dry for 1/2 hr. No longer. Excessive drying may oxidize
the fat and give high results.
- Cool in a desiccator and weigh and record weight to the
nearest 0.1 mg (W2).
- The fat beakers are best cleaned by warming on a
steambath or on a hot plate on a low setting. Add some
used ether to dissolve the fat. The use of a rubber
policeman is helpful. After soaking the beakers in
Alconox detergent, wash them using hot water and vigorous
brushing. The thimbles are best cleaned by blowing out
- If doing a proximate analysis, the residue left in the
thimble may be used to determine crude fiber.
Calculations: Percent Crude Fat (Ether Extract), DM
% Crude Fat (DM basis) = (W3 - W2) X 100 / W1
X Lab DM / 100
- W1 = initial sample weight in grams
- W2 = tare weight of beaker in grams
- W3 = weight of beaker and fat residue in grams
Include a reagent blank and one or more quality control (QC)
samples in each run, choosing QC samples by matching analyte
levels and matrices of QC samples to the samples in the run.
Include at least one set of duplicates in each run if single
determinations are being made.
An acceptable average standard deviation among replicated
analyses for crude fat about ±0.10, which results in a warning
limit (2s) of ±0.20 and a control limit (3s) of ±0.30. Plot the
results of the control sample(s) on an X-control chart and
examine the chart for trends. Results outside of upper or lower
warning limits, ±2 standard deviations (95 percent confidence
limits), are evidence of possible problems with the analytical
system. Results outside of upper or lower control limits, ±3
standard deviations (99 percent confidence limits), indicate loss
of control, and results of the run should be discarded. Two
consecutive analyses falling on one side of the mean between the
warning limits and the control limits also indicate loss of