Standard Operating Procedure
TO BE PERFORMED AFTER ACID DETERGENT FIBER ANALYSIS
Title: Acid Detergent Lignin Analysis via Beaker Technique with Ashing
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 Lignin, which is the residue
remaining after digesting with concentrated sulfuric acid (H2SO4). The remaining fiber
residues are predominantly cellulose and lignin.
Equipment and reagents necessary:
Reagents:
Sulfuric Acid (72% by weight, purchased pre-standardized from ANKOM Technology)
Acetone
Miscellaneous:
2L and 3L beaker
Oven @ 105 ˚C
Waste container for acid waste
MoistureStop weigh pouch
Analytical balance
Crucibles
Muffle furnace, Trish Patrick’s lab
Litmus paper for measuring pH
Procedure:
1. Perform NDF determination using ANKOM Fiber Analyzer (see Neutral Detergent Fiber
procedure)
2. Perform ADF determination using ANKOM Fiber Analyzer (see Acid Detergent Fiber
procedure)
3. IMPORTANT: Ensure bags are completely dry and are at ambient temperature before
adding acid. If moisture is present in the bags, heat generated by the sulfuric acid –
water reaction will adversely affect the results.
4. Place dried bags into 3L beaker and add sufficient quantity (~250 ml) of 72% H2SO4 to
cover bags.
5. Place 2L beaker inside 3L beaker to keep bags submerged. Agitate bags at start and at
30 minute intervals by gently pushing and lifting 2L beaker ~30 times.
6. After 3 hours pour off 72% H2SO4 into waste container and rinse bags with tap water to
remove all acid. Repeat rinses until pH is neutral, confirmed using Litmus paper. Rinse
with ~250 ml acetone for 3 minutes to remove water. Allow acetone to evaporate.
7. DO NOT place bags in oven until acetone is completely evaporated, as acetone is
extremely flammable.
8. Complete drying in oven at 105°C for 2-4 hours.
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9. Remove bags from oven and place directly in MoistureStop weigh pouch, flattening to
remove air.
10. Cool to ambient temperature and weigh (W 3).
11. Ash entire bag in pre-weighed crucible (30-50 ml) at 525°C for 3 hours or until C-free.
Cool and calculate weight loss (W 4). Calculate blank bag ash correction (C2) using
weight loss upon ignition of a blank bag sequentially run through ADF and lignin steps.
Calculations
ADL (as received basis) =[ [(W3 – (W1 x C1))] / W2 ] x 100
ADLDM (DM basis) =[ [(W3 – (W1 x C1))] / (W2 x DM) ] x 100
ADLOM (as received basis) =[ [(W4 – (W1 x C2))] / (W2 x DM)] x 100
W1 = bag tare weight
W2 = sample weight
W3 = dried weight of bag with fiber after extraction process
W4 = weight of organic matter (loss of weight on ignition of bag and fiber residue)
C1 = blank bag correction
(final oven weight of blank bag) / (original blank bag weight)
C2 = Ash corrected blank bag
(loss of weight on ignition of bag) / (original blank bag weight)
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:
Sulfuric Acid, 72%: 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.
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Spill/Release Containment and Clean Up/Decontamination Procedures:
Sulfuric Acid, 72%: 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:
Sulfuric Acid, 72% 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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 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.
Sulfuric Acid, 72% MSDS, available through ANKOM website.
Acetone MSDS, available through ANKOM website
Dec 2012 VNichols