Standard Operating Procedure
Title: Acetyl Bromide Methods for Lignin Extraction
Department: Agronomy
Created by: Mark Westgate
Revised by: Andrea Rouse
Laboratory: Crop Production & Physiology
Lab Supervisor: Maria Hartt Eckerman me
Date approved: 11Feb2005
Procedure Overview: This procedure is used quantify total lignin in plant tissues.
Equipment and reagents necessary:
Plant Tissue:
soybean seedlings, 7-10 days after planting
Reagents (all chemicals are reagent grade): Amounts listed are for stock solutions so that
multiple samples can be run.
Liquid nitrogen
Phosphorous pentoxide desiccant crystals (use silica gel or Drierite)
80% ethanol (1.5 L)
Chloroform (167 ml)
Methanol (83 ml)
Acetone (250 ml)
10 mM potassium phosphate, monobasic (pH 6.0) (500 ml)
α-amylase, type XII-A from Bacillus licheniformis (EC 3.2.1.1; Sigma A-3403, 1 IU mg-1) (1 unit per
sample)
Amyloglucosidase from Aspergillus niger (EC 3.2.1.3. Fluka; 50 IU mg-1) (1 unit per sample)
Deionized water
Acetyl bromide reagent (25% v/v acetyl bromide in glacial acetic acid) (4 ml per sample made fresh
before use)
0.5 M hydroxylamine solution (250 ml)
Acetic acid, glacial (750 ml)
2 M sodium hydroxide (500ml)
NIST Eastern cottonwood standard (SRM #8492) (100-200mg)
Equipment:
2 L plastic Nalgene beaker
Labconco freeze dryer
Wiley mill
mg scale
Spatula
Weigh paper/boats
pH meter
Miscellaneous:
Razor blades
50 mL polypropylene tubes
Capped glass culture tubes (16 x 150 mm)
96-well plates
Sonicator
Table-top centrifuge
55ºC water bath
50ºC dry block heater
Vacuum desiccator
Scanning spectrophotometer
5 mL pipettes & tips
50 mL volumetric flasks
10 & 50 mL graduated cylinders
04may04 mew
Revised 10feb05 adr
Procedure:
Tissue preparation: (soybean seedlings 7-10 days after planting)
1. Wash plant tissue with tap water until free of soil debris.
2. Using a razor blade, separate plants into stem, root, cotyledon, and apex. Bulk
samples from 10 plants and place in labeled 50 mL conical tubes
3. Freeze tissues by briefly placing tubes in plastic Nalgene beaker containing liquid
nitrogen. While wearing protective gloves &/or using tongs, remove tubes and place
in -80C freezer until samples are ready to be lyophilized.
4. Freeze-dry plant tissues for 48 h on Labconco freeze dryer (follow Freeze Drier
Operation SOP).
5. Using a Wiley Mill, grind plant tissues to pass a 0.5 mm sieve.
6. Place ground samples in labeled screw-cap vials and store samples in a desiccator
at room temperature.
Cell Wall preparation:
1. For each sample, weigh 100-200 mg of lyophilized plant tissue; transfer to properly
labeled 50 mL polypropylene tube.
2. Working in the hood, extract plant tissues (100-200 mg) with four sequential
sonications in 80% v/v ethanol (40 mL g-1, 10 min) following each with centrifugation
(3000g, 15 min). Discard supernatant in appropriately marked waste container.
3. Extract residue with one sonication in chloroform/methanol (2:1, 40 mL g -1, 2 min).
Centrifuge at 3000g as needed to pellet residue. Discard supernatant in
appropriately marked waste container.
4. Extract residue with one sonication in acetone (40 mL g-1, 2 min). Centrifuge at
3000g as needed to pellet residue. Discard supernatant in appropriately marked
waste container.
5. Air dry Cell Wall (CW) residue.
6. Suspend air-dried CW material in 10 mM potassium phosphate buffer (pH 6.0, 30 mL
g-1).
7. Heat tubes in water bath at 90ºC for 90 minutes to gelatinize starch.
8. Cool tubes to 55ºC.
9. Digest CW starch with α-amylase (Sigma A3403 from Bacillus) and
amyloglucosidase (Fluka 10115 from Aspergillus) as follows:
a. Add 10 units of α-amylase to each tube; mix thoroughly and incubate in a
water bath at 55ºC for 90 minutes
b. Adjust pH to 6.0.
c. Add 10 units of amyloglucosidase to each tube; mix thoroughly and incubate
in a water bath at 55ºC for 60 minutes.
10. Centrifuge CW residue at 3000g for 15 min.
11. NOTE: The pellets will be very loose after step 10. Take extra care not to lose the
pellet during the following washing steps. Wash with 30 mL deionized water.
Centrifuge at 3000g for 15 min.; discard supernatant in appropriately marked waste
container.
12. Repeat washing (step 11) three times.
13. Freeze CW pellet in -80C then lyophilize. Store dried CW over desiccant.
14. This procedure should yield lignin extraction percents of over 98%. Typical lignin
absorbancies should be approximately 0.001 to 0.2 above background. Typical
standard curve slopes should be between 0.4 and 0.6 conc/abs.
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04may04 mew
Revised 10feb05 adr
Lignin Extraction:
1. Transfer 100 mg of freeze-dried CW to glass culture tubes (16 x 150 mm) fitted with
Teflon-lined screw caps.
2. Working in a hood, add 4.0 mL of freshly prepared acetyl bromide reagent (25% v/v
acetyl bromide in glacial acetic acid) to each tube; cap immediately.
3. Heat tubes in dry block (in 1522 hood) at 50ºC for 2-4 h with occasional mixing.
Incubation of 4 hours will result in over 98% lignin extraction.
4. Cool samples in hood. Bring volume to 15 mL with acetic acid; pellet residue at
3000g for 15 min.
Lignin Quantification:
1. Working in the hood, to a tube containing 2.5 mL of acetic acid add 0.5 mL of the
lignin extract and 1.5 mL of 0.3 M NaOH.
2. After mixing, add 0.5 mL of 0.5 M hydroxylamine (in hood) and dilute to 10 mL with
acetic acid.
3. Transfer 250 μL of each sample into a well of a 96-well microreader plate. Include a
blank (acetic acid + hydroxylamine) to correct for reagent background.
4. Measure absorbance within 30 min at 280 nm in the microplate scanning
spectrophotometer (1522).
5. Calculate lignin concentration as C (g L-1) = cm x 17 x A280 where cm is the path
length of the instrument. This calculation can be converted to grams of lignin by
comparing the A280 reading for a sample to the lignin standard for Eastern
Cottonwood (NIST #8492).
Personal Protective Equipment / Engineering Controls:
Eye protection (goggles & face shield)
Skin protection (proper shoes, gloves, lab coat, etc.)
Ventilation system/ Reach-in hood
Safety shower
Eye wash station
Hazard Controls & Storage Precautions:
Acetic acid, glacial: Use under fume hood. Store in tightly closed container away from
incompatibles, heat, sparks, and flame.
Acetone: Store in tightly closed container away from incompatibles, heat, sparks, and
flame.
Acetyl bromide: Use only in fume hood. Wash after handling. Store under nitrogen in
tightly closed container. Reacts violently with water.
-amylase: Mechanical exhaust required. Store tightly closed at 2-8C.
Amyloglucosidase: Store at 2-8C in tightly closed container away from incompatibles.
Avoid inhalation.
Chloroform: Potential carcinogen. Has caused adverse reproductive and fetal
effects in animals. Store in tightly closed container away from incompatibles, heat,
sparks, and flame. Light sensitive.
Ethanol: Store in tightly closed container away from incompatibles, heat, sparks, and
flame. Do not expose to direct sunlight.
Hydroxylamine: Dangerous to the environment. Heating may cause an explosion. Use
only under fume hood with appropriate PPE. Store in tightly closed container away
from incompatibles, heat, sparks, and flame.
Methanol: Poison!
Cannot be made non-poisonous.
Has caused adverse
reproductive and fetal effects in animals. Use exhaust ventilation equipment.
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04may04 mew
Revised 10feb05 adr
Store in tightly closed container away from incompatibles, heat, sparks, and flame.
Empty containers retain product residue.
Nitrogen, liquid: Can cause frostbite; wear protective gloves. Asphyxiant.
Potassium phosphate, monobasic: Hygroscopic. Store in tightly closed container,
protected from moisture.
Silica gel: Use with adequate ventilation. Keep container tightly closed.
Sodium hydroxide: Corrosive. Use under fume hood. Hygroscopic; avoid adding water
to material. Store in tightly closed container away from incompatibles.
Waste Disposal & Decontamination Procedures:
Acetic Acid, Glacial: Place with compatible solutes in tightly closed designated waste
container in Hazardous Waste Satellite Accumulation Area.
Acetone: Place with compatible solutes in tightly closed designated waste container in
Hazardous Waste Satellite Accumulation Area.
Acetyl bromide: Place with compatible solutes in tightly closed designated waste
container in Hazardous Waste Satellite Accumulation Area.
-amylase: Place with compatible solutes in tightly closed designated waste container in
Hazardous Waste Satellite Accumulation Area.
Amyloglucosidase: Place with compatible solutes in tightly closed designated waste
container in Hazardous Waste Satellite Accumulation Area.
Chloroform: Place with compatible solutes in tightly closed designated waste container
in Hazardous Waste Satellite Accumulation Area.
Ethanol: Place with compatible solutes in tightly closed designated waste container in
Hazardous Waste Satellite Accumulation Area.
Hydroxylamine: Place with compatible solutes in tightly closed designated waste
container in Hazardous Waste Satellite Accumulation Area.
Methanol: Place with compatible solutes in tightly closed designated waste container in
Hazardous Waste Satellite Accumulation Area.
Nitrogen, liquid: Allow to evaporate. Do not pour in sink as sink will crack.
Potassium phosphate, monobasic: Place with compatible solutes in tightly closed
designated waste container in Hazardous Waste Satellite Accumulation Area.
Silica gel: Place with compatible solutes in tightly closed designated waste container in
Hazardous Waste Satellite Accumulation Area.
Sodium hydroxide: Place with compatible solutes in tightly closed designated waste
container in Hazardous Waste Satellite Accumulation Area.
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04may04 mew
Revised 10feb05 adr
Health & Safety Info for Required Reagents:
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Chemical name
Acetic Acid, glacial
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Y
Acetyl Bromide
Ethanol
Y
Hydroxylamine
Y Y
Y Y
Y Y
Y
Y Y
Y Y
Y
Sodium hydroxide
Y
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Kidneys, heart,
CNS, liver, eyes
Y
Y Y
Y
Eyes, skin
Y
none
Y
Eyes, skin,
mucous
membrane,
respiratory tract
Incompatibilities
Y Y
Avoid high temperatures,
ignition sources,
strong oxidizing agents
and strong acids
Water, strong oxidizing
agents, strong bases,
alcohols.
Oxidizing agents
1 3 0
Y
Y
Y
Strong oxidizing agents
High temperatures, light,
strong oxidizing agents,
aluminum, fluorine,
magnesium, sodium,
potassium, lithium,
caustics, ditrogen
tetraoxide
Oxidizing materials,
peroxides, acids, acid
chlorides, acid
anhydrides, alkali metals,
ammonia, moisture,
aluminum acetyl chloride,
strong reducing agents
Oxidizing agents,
potassium dichromate,
chromium trioxide, zinc,
calcium, copper, sodium,
ammonia, phosphorus
halides, carbonyls,
pyridine, hypochlorites.
Avoid high temperatures,
ignition sources,
strong oxidizing agents,
strong acids, aliphatic
amines, caustics
Under certain conditions:
lithium, neodymium,
titanium, ozone
strong oxidizing agents,
strong bases.
Fluorides, hydrochloric
acid, strong oxidizers,
vinyl acetate.
Water, metals, acids,
aluminum, zinc, tin,
nitrometane, leather,
flammable liquids, organic
halogens, wool.
The above summary consists of guidelines for proper handling & disposal
of chemicals used in this procedure. Please read attached MSDSs for more
specific information.
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3 2 0
Blood, central
nervous system,
mucous
membranes
Y
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y
Avoid oxidizing materials,
and alkaline substances.
Y
Y
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Y
skin
Nitrogen, liquid
Potassium
phosphate
Silica gel
desiccant
F
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m
m
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Y
Eyes, skin
Blood, kidneys,
heart, CNS, liver,
cardiovascular
system, excretory
system,
reproductive
system
Kidneys, heart,
CNS, liver
Y Y Y Y Y
Methanol
E
x
p
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e
Eyes, skin
mucous
membranes
Eyes, skin
Y Y Y
Y Y
C
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G
a
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Teeth, eyes, skin,
mucous
membranes
CNS, respiratory
system, eyes,
skin
Y
Y Y
-amylase
Amyloglucosidase
Chloroform
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Target Organ
Acetone

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04may04 mew
Revised 10feb05 adr
3 2 2
3 0 0
1 0 0
2 0 0
1 3 0
3 3 0
1 3 0
3 0 0
1 0 0
1 0 0
3 0 1