Standard Operating Procedure
TO BE PERFORMED AFTER NEUTRAL DETERGENT FIBER ANALYSIS
Title: Acid Detergent Fiber Analysis via Filter Bag Technique for samples
containing < 5% fat (no soybean products)
Department: Agronomy
Created by: Virginia Nichols
Laboratory: Crop Production & Physiology Lab Suite
Supervisor: Dr. Fernando Miguez
Lab Supervisor: Whitney Bouma
Date approved:
Procedure Overview: This procedure determines Acid Detergent Fiber, which is the residue
remaining after digesting with sulfuric acid (H2SO) and hexadecyl-trimethyl-ammonium
bromide (CTAB). The remaining fiber residues are predominantly cellulose and lignin.
Equipment and reagents necessary:
Reagents:
Acid Detergent (AD) Solution (2000 mL per batch)
purchase pre-mixed from ANKOM Technology
2.93% H2SO4 concentrate
2.00% CTAB
balance water
Acetone
Water
Miscellaneous:
Analytical Balance (0.1 mg accuracy)
Weighing spatulas
Oven (capable of maintaining a temperature of 102±2⁰C)
ANKOM 200 Digestion instrument
Chemically inert filter bags (purchased from ANKOM Technology)
Heat sealer (purchased from ANKOM)
Desiccator pouch
Solvent and acid resistant marking pen
Appropriate waste vessel (2)
Gloves
Lab coat
Safety glasses
Procedure:
1. Perform NDF determination using ANKOM Fiber Analyzer (see Neutral Detergent Fiber
procedure)
2. Use the same samples for all subsequent steps.
3. Place no more than 24 bags into the Bag Suspender. 8 trays should be used regardless
of the number of bags being processed – the 9th tray is used as a cover and should NOT
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have bags in it. Place no more than 3 bags per tray, then stack trays on center post with
each level rotated 120 degrees.
4. Insert the Bag Suspender with bags into the fiber analyzer vessel and place the weight
on top to keep it submerged.
5. The amount of AD solution added will depend on the number of bags you use.

When processing 20-24 bags, add 1900 – 2000 mL of ambient AD solution to the
fiber analyzer vessel.

When processing less than 20 bags, add 100 mL/bag of AD solution, with a
minimum of 1500 mL to ensure the bag suspender is covered.
6. Make sure all switches are ‘off’ before plugging in in the instrument. Once plugged in,
turn Agitate and Heat on and confirm agitation. Set timer for 60 min and close lid. Vessel
will maintain a solution temperature of 100⁰C
7. At the end of the extraction, turn Heat and Agitate off. Open the drain valve (slowly!) and
drain the AD solution into a waste vessel before on before opening lid. Note that the
exhaust hose must be secured to the vessel, as the contents are under pressure.
8. (Rinse 1) After the solution has been drained, close the valve and open the lid. Add 1900
mL of (70-90°C) tap water. Turn Agitate on and rinse for 5 min. The lid may be sealed
with Heat on or left open with Heat off. Drain the solution into a container – it is your
decision whether or not this rinse is considered waste.
9. (Rinse 2) Repeat step 8.
10. (Rinse 3) Repeat step 8.
11. Remove the samples, and gently press out excess water from bags.
12. Place bags in a 250 mL beaker, add enough acetone to cover bags and soak for 3-5
min.
13. Remove bags from acetone and place on wire screen to air dry. Completely dry in oven
at 102 ± 2°C. Most ovens will complete drying within 2-4 hours. Do NOT place bags in
oven until acetone has completely evaporated (as acetone is flammable).
14. Remove bags from oven, place directly into a collapsible desiccant pouch and flatten to
remove air. Do NOT use a conventional desiccator container. Cool to ambient
temperature.
15. Weigh bags (W 3). Blank bag correction (C1) is the final oven-dried weight divided by the
original blank bag weight.
16. If vessel will not be used for >1 month, add 1900 mL of water, agitate 10 min, drain. This
will ensure there is no acid remaining to corrode the machine.
Calculations
% ADF (as received basis) =[ [(W3 – (W1 x C1))] / W2 ] x 100
W1 = bag tare weight
W2 = sample weight
W3 = dried weight of bag with fiber after extraction process
C1 = (final oven weight of blank bag) / (original blank bag weight)
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Personal Protective Equipment / Engineering Controls:
Nitrile gloves
Safety glasses
Face shield
Dust mask
Latex gloves
Splash goggles
Lab coat
Fume hood
Neoprene gloves
Vented goggles
Apron
Biosafety cabinet
Insulated gloves
Eye wash station
Safety shower
Respirator
Note: Open-toed and heeled shoes are NOT allowed.
Other Control Measures:
Handling & Storage Precautions:
Acid Detergent (AD) Solution: Use with adequate ventilation, wash hands and clothing thoroughly after
handling. Store in a cool, dry, well-ventilated area away from incompatible substances. Never add
water to this solution – always add acid slowly and in small amounts to water.
Acetone: Use with adequate ventilation, wash hands and clothing thoroughly after handling. Store in a
cool, dry, well-ventilated area away from incompatible substances and away from ignition sources.
Waste Disposal Procedures:
Unless EH&S specifically instructs otherwise, all chemical/reagent waste (including excess solutions)
must be placed in an appropriately labeled hazardous waste container for EH&S disposal.
Compatible substances may be combined into one waste container.
Spill/Release Containment and Clean Up/Decontamination Procedures:
Acid Detergent (AD) Solution: Soak up with an inert absorbent material. Rinse area with water.
Acetone: Remove all sources of ignition and provide adequate ventilation. Soak up with an inert
absorbent material.
Health & Safety Summary for Required Reagents:
The Acid Detergent (AD) Solution is a stable, non-flammable, corrosive substance. It should not be
heated above 250°C. Use extreme caution not to allow solution to contact skin. It is a moderate
health risk and a severe contact risk. DO NOT add water to this solution.
Acetone is flammable – avoid excessive temperatures, heat, sparks, open flame, and other sources of
ignition. It has a very high vapor pressure, and should therefore only be used in well-ventilated
areas.
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Chemical name
Sodium sulfite
Acid Detergent
Solution
-H2SO4 (25%)
- CTAB (10%)
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Target Organ(s)
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Eyes, skin, teeth
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Eyes, skin, teeth
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Alkalies, amines, anhydrides,
combustibles, organics,
oxidizers, powdered
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Incompatibilities
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Acetone
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Central
system
nervous
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Chloroform, chromic
anhydride, hydrogen
peroxide, nitric
compounds, acids, strong
oxidizers, alkalies.
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 The above summary consists of guidelines for proper handling & disposal of
chemicals used in this procedure. You must read and understand the
contents of the entire MSDS(s) before starting this procedure.
References:
ANKOM Procedures. available through ANKOM website.
Acid Detergent Solution MSDS. available through ANKOM website.
Acetone MSDS, available through ANKOM website
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