4.1 Determination of Acid Detergent Fiber by Refluxing
Fiber (Acid Detergent) and Lignin in Animal Feed. (973.18)
Official Methods of Analysis. 1990. Association of Official
Analytical Chemists. 15th Edition.
This procedure is applicable for the determination of acid
detergent fiber (ADF) in all types of forages.
An acidified quaternary detergent solution is used to dissolve
cell solubles, hemicellulose and soluble minerals leaving a
residue of cellulose, lignin, and heat damaged protein and a
portion of cell wall protein and minerals (ash). ADF is
determined gravimetrically as the residue remaining after
- Refluxing apparatus
- Berzelius beakers (600 mL)
- Fritted glass (Gooch) crucibles (coarse porosity, 50 mL)
- Analytical electronic balance, accurate to 0.1 mg
- Suction filtering device with trap in line and valve to
- Forced-air drying oven set at 100oC
Acid detergent solution:
1 liter of 1.00N Sulfuric acid ±0.005N. Normality must be
verified by titration with a primary base standard (method 3.1.2)
before adding CTAB. A solution approximately 1.0 N sulfuric acid
can be made by adding 51.04 g (27.7 mL) of concentrated reagent
grade sulfuric acid (95-98% purity) to 972.3 mL water (AOAC
935.70). Titrate by method 3.1.2 and add water (if
normality too high) or sulfuric acid (if normality too low) to
adjust normality to 1.00N ±0.005N. 20 g Cetyl trimethylammonium
bromide (CTAB), technical grade Acetone, reagent grade
- Always add sulfuric acid to water. Wear face shield and
heavy rubber gloves. If acid is splashed on skin, wash
immediately with copius amounts of water.
- CTAB powder will irritate mucous membranes, eyes and
skin. Wear gloves and dust mask while handling.
- Acetone is highly flammable. Do not let vapors accumulate
in work area. Use effective fume removal device. Also
avoid inhaling or contact with skin. Make sure all traces
of acetone have evaporated from the crucibles containing
fiber residue before placing in the drying oven.
- Samples should be microwave dried or oven dried at 55oC
to ³85% dry matter, then ground to pass a 1 mm screen.
- Dry 50 mL fritted glass crucibles overnight at 100oC and
hot weigh (W1), recording weight to nearest 0.1 mg. (Hot
weigh techniques described in method
- Thoroughly mix and weigh sample (W2) (approximately 0.9
to 1.1 g, record weight accurate to 0.1 mg) into
Berzelius beaker. Weigh a second subsample for laboratory
dry matter determination.
- Add 100 mL acid-detergent solution at room temperature.
Place beaker on heater under the cold water condenser.
- Heat to boiling in 5-10 min; reduce heat to avoid foaming
as boiling begins. Reflux 60 min from onset of boil,
adjusting boiling to slow, even level.
- After about 30 min, wash down sides of beaker with
minimal amount of acid detergent solution. A wash bottle
is convenient for dispensing solution.
- Remove beaker, swirl, and filter through tared (step 2)
fritted glass crucible, using minimal vacuum. Police and
rinse the Berzelius beaker with boiling water while
inverted over the crucible to insure quantitative
transfer of all fiber particles into the crucible.
- Soak twice with boiling (95-100oC) water by breaking up
mat and filling crucible each time with vacuum off and
allowing to soak a minimum of 15 to 30 sec (2 min
recommended) after each wash. While filling the crucible
with hot water or acetone, rinse the top edge and sides
to remove residual acid detergent.
- Rinse twice with 30-40 mL acetone by filling crucible
each time with vacuum off, allowing a minimum of 15 to 30
sec (2 min recommended) before vacuuming dry.
- Dry 3 hr or overnight in forced-air oven (100oC) and
weigh hot, recording weight (W3) to nearest 0.1 mg.
- Sulfuric acid for acid detergent fiber solution must be
standardized to be between 0.995 and 1.005 N. Variation
in normality outside of this range can result in low or
high ADF values.
- Timing of refluxing is critical and should not vary more
than 5 min from the 60 min described by the method.
- Acid must be thoroughly washed from the sample because it
will become concentrated when water is removed during
drying. The combination of strong sulfuric acid and high
temperature can char the sample and result in low ADF
values. If black discoloration occurs during drying,
repeat the analysis.
- Difficult filtration may result from plugging of the
fritted glass crucibles. Crucibles should be cleaned
regularly with acid or alkaline cleaning solution.
(Alkali cleaning will tend to deteriorate fritted disk
faster.) The filtration rate of crucibles should be as
uniform as possible for a given set of samples. To check
the filtration rate of crucibles, fill them with 50 mL of
distilled water and record the time required to drain
completely without vacuum. This should be about 180 sec.
If filtration takes more than 240 sec, crucibles need
cleaning. If cleaning does not improve the filtration
rate, the crucible should be discarded. If filtering
takes less than 120 sec, check crucible for cracks or
holes in the fritted disk. If filtering takes less than
100 sec, the crucible should be discarded.
- The proper vacuum is critical to good filtering. It
should be sufficient to remove the solutions rapidly but
not so great that fiber particles plug the fritted disk.
- Rinse water must be in excess of 95oC. This is
particularly true of samples containing pectic
substances, mucilages or glycoproteins.
Calculation: Percent Acid Detergent Fiber (ADF)
% ADF (DM basis) = (W3 - W1 / W2 X Lab DM /
100) X 100
- W1 = tare weight of crucible in grams
- W2 = initial sample weight in grams
- W3 = dry weight of crucible and dry fiber in grams
Include 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 acid detergent fiber ranges from about ±0.20 for
samples with 20% ADF to ±0.35 for samples with 40% ADF, which
results in warning limits (2s) ranging from ±0.40 to 0.70 and
control limits (3s) ranging from ±0.60 to 1.05. 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, ±2s (95 percent confidence limits), are evidence of
possible problems with the analytical system. Results outside of
upper or lower control limits, ±3s (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 control.