Ankom Protocol

The Ankom Fiber Analyzer utilizes a series of extractions to determine the fiber content of a plant sample. Each of the extractions should be done in the order of NDF (Neutral Detergent Fiber), ADF (Acid Detergent Fiber), and then ADL (Acid Determined Lignin). The NDF solution is just soapy water. During NDF, the fraction that is washed off contains soluble cell contents like carbohydrates, lipids, pectin, starch, soluble proteins and non-protein nitrogen. The fraction that is left in the bag contains hemicellulose, proteins bound to the cell walls, cellulose, lignin, and recalcitrant materials. ADF solution is a 1.00 Normal sulfuric acid and detergent solution. During ADF, hemicellulose and bound proteins are washed off. The fraction left behind contains cellulose, lignin, and recalcitrant materials. The ADL solution is ultra nasty 72% sulfuric acid. During ADL, cellulose is washed off leaving only lignin and recalcitrant materials.

MATERIALS

  • Ankom fiber analyzer
  • Neutral Detergent Solution
  • Acid Detergent 5x solution
  • 72% Sulfuric acid
  • Pure Acetone
  • Acid resistant gloves
  • Acetone resistant gloves
  • Filter bags
  • Heat sealer
  • Ankom marker (acid resistant)
  • Glass trays
  • 3-4 Aluminum pie tins
  • 2-3 Electric tea kettles
  • Muffle furnace
  • Crucibles
  • Crucible tongs

SAFETY PRECAUTIONS

  • Acetone is highly flammable. Use fume hood when handling acetone and avoid inhaling or contact with skin (wear lab coat, safety glasses and green acetone resistant gloves). Make sure bags are completely dry and that all the acetone has evaporated before placing in oven.
  • Blue acid resistance rubber gloves, lab coat and safety glasses should be worn when handling sulfuric acid. Always add sulfuric acid to water. If acid contacts skin wash with copious amounts of water.
  • For more detailed information please see material safety data sheets.

METHODS

Day 1:

Sample Prep

  1. Grind samples using a 40mm screen and air dry.
  2. Label filter bags with special Ankom marker or Sharpie (Note: Sharpie will eventually fade and will need to be relabeled)
  3. Dry bags in oven at 105C for 30 min. Cool in dessicator for 30 min.
  4. Weigh each bag alone and tare balance.
  5. Weigh out .5 g ( 0.05 g) of sample into bag and record weight. Include a blank bag in each run (each batch of 24) for a blank bag correction.
  6. Seal the bag closed within 0.5 cm from the open edge using the heat sealer. Be sure to also seal the blank bag.
  7. Spread sample uniformly inside the filter bag by shaking and lightly flicking the bag to eliminate clumping.
  8. A maximum of 24 bags may be placed in the sample tree. All nine trays are used regardless of the number of bags being processed. Place three bags per tray and then stack trays on center post with each level rotated 120 degrees. Each tray will sit in the notches of the tray below it. The weight is placed on top of the empty 9th tray to keep the bag suspender submerged.

NDF Extraction: This method determines Neutral Detergent Fiber, which is the residue remaining after digesting in adetergent solution. The fiber residues are predominantly hemicelluloses, cellulose, and lignin.

  1. Place bag suspender into the chamber with weight.
  2. When processing 24 sample bags add 1800-1900 ml of ambient Neutral Detergent solution into the chamber. Be sure the valve on the left side of the machine is closed! If you’re processing less than 24 samples, load the bag suspender from the bottom up and fill the chamber until the bags are covered. I found that 13 bags are covered by 1500 ml of solution.
  3. Turn Agitate and Heat ON and confirm that the bag suspender is agitating properly. Set the timer for 75 minutes. Close and seal the lid of the chamber.
  4. Fill 2-3 teakettles with DI water and start them boiling 10 minutes before the end of the run.
  5. After 75 minutes turn Agitate and Heat OFF, open the drain valve and drain the hot solution BEFORE opening the lid. The neutral detergent can be dumped down the drain. WARNINING: The solution in the vessel is under pressure. The valve should be opened first to remove pressure before the lid can be opened. Ensure that the exhaust hose is securely positioned for safe disposal of effluent.
  6. After solution has been exhausted, close the valve and open the lid. Pour in rinse #1 of boiling water to ~2 cm below lip of chamber. Lower lid but do not seal. Agitate for 5 minutes, but do NOT Heat. Refill kettle.
  7. After 5 minutes, drain the rinse water down the sink. Repeat rinse 3 more times for a total of 4 rinses.
  8. Remove the tree stand and place in a plastic tub. With acetone gloves, gently press water from bags and place in 3 or 4 L beaker. Add enough acetone to cover bags and soak for 3 minutes, gently agitating.
  9. After 3 minutes, pour out acetone into a waste container. With acetone gloves, gently press acetone out of bags. Spread bags out on glass baking dishes in hood and allow to dry for ~ 40 minutes.
  10. Place bags in an aluminum cake pan and dry at 105C overnight.

WARNING: Do not place bags in oven until acetone has completely evaporated

Day 2:

ADF Extractions: This method determines Acid Detergent Fiber, which is the residue remaining after digesting with H2SO4 and CTAB. The fiber residues are predominantly cellulose and lignin.

  1. Remove dried bags from oven and cool in desiccator for 30 minutes. Weigh dried bags and replace bags in the tree.
  2. Add 1800-1900 ml of ADF solution into the chamber. Be sure the valve on the left side of the machine is closed!
  3. Turn Agitate and Heat ON and confirm that the bag suspender is agitating properly. Set the timer for 60 minutes. Close and seal the lid of the chamber.
  4. Fill 2-3 teakettles with DI water and start them boiling 10 minutes before the end of the run.
  5. After 60 minutes turn Agitate and Heat OFF, open the drain valve and drain the hot solution BEFORE opening the lid. The solution can be dumped down the drain. WARNINING: The solution in the vessel is under pressure. The valve should be opened first to remove pressure before the lid can be opened. Ensure that the exhaust hose is securely positioned for safe disposal of effluent.
  6. After solution has been exhausted, close the valve and open the lid. Pour in rinse #1 of boiling water to ~2 cm below lip of chamber. Lower lid but do not seal. Agitate for 5 minutes, but do NOT Heat. Refill kettle.
  7. After 5 minutes, drain the rinse water down the sink. Repeat rinse 3 more times for a total of 4 rinses.
  8. Remove the tree stand and place in a plastic tub. With acetone gloves, gently press water from bags and place in 3 or 4 L beaker. Add enough acetone to cover bags and soak for 3 minutes, gently agitating.
  9. After 3 minutes, pour out acetone into a waste container. With acetone gloves, gently press acetone out of bags. Spread bags out on glass baking dishes in hood and allow to dry for ~ 40 minutes.
  10. Place bags in an aluminum cake pan and dry at 105C overnight.

WARNING: Do not place bags in oven until acetone has completely evaporated

Day 3:

ADL Extraction

  1. Remove dried bags from oven and cool in desiccator for 30 minutes. Weigh dried bags and place them into a 3 L beaker.
  2. Wearing safety goggles and acid resistant gloves, add enough sulfuric acid (72% H2SO4) to cover bags (~300 ml). It is important that the bags are completely dry. If there is any water in the sample, the reaction of the water with the strong sulfuric acid will generate heat. The sample could char and you could lose sample.
  3. Place a 2 L beaker inside the 3 L beaker to keep bags submerged. Agitate bags at start and at 30-minute intervals for 3 hours. Agitate by lifting the 2L beaker up and down ~ 30 times.
  4. Fill 2-3 kettles with DI water and start boiling 10 minutes before the end of the run.
  5. Wearing safety goggles and acid resistant gloves, pour off sulfuric acid into waste container. Fill the beaker with boiling water and rinse for 5 minutes. Place 2 L beaker inside of 3 L beaker to keep bags submerged and to agitate. After 5 minutes discard rinse water down the drain with lots of water.
  6. Repeat the rinse 3 more times for a total of 4 rinses.
  7. With acetone gloves, gently press water from bags and place in a 3 or 4 L beaker. Add enough acetone to cover bags and soak for 3 minutes, gently agitating.
  8. Pour off acetone into waste container. With acetone gloves, gently press acetone out of bags and spread out on baking dishes in the hood to dry (~40 min).
  9. Place bags in aluminum cake pan and dry at 105C overnight.

WARNING: Do not place bags in oven until acetone has completely evaporated

72% H2SO4

Materials:

  • 2 liter flask
  • glass beaker
  • face shield
  • acid resistant gloves
  • lab coat and apron
  • balance
  • ice bath

Method:

  1. Place beaker on scale and tare weight.
  2. Using beaker weight out 829 grams of DI water to flask.
  3. Place flask in ice bath.
  4. In ice bath with full protection slowly add 2439 grams H2SO4. Add small portions and stop to swirl in ice bath to cool between portions.
  5. Let cool to room temperature.
  6. Use either weight (3268 g) or read within 0.5 cm of line.

ASHING

  1. Rinse crucibles with DI water and dry at 105C overnight.
  2. Turn muffle furnace on to 150C and let sit overnight. Make sure the hood vent in room 418 is also turned on.

Day 4:

ASHING (cont’d)

  1. Remove crucibles from the oven and place in the dessicator for 1 hour. NOTE: Do not touch crucibles with bare hands once they have been cleaned and dried. Use gloves or crucible tongs. After drying, weigh crucibles and record the number.
  2. Remove dried bags from oven and cool in desiccator for 30 minutes. Weigh bags and record the weight.
  3. Fold bags first lengthwise and then again width wise and place in crucibles rounded part up.
  4. Place in muffle furnace at 500C for 5.5 hours. After 5.5 hours turn down temperature to 150C and leave samples in furnace overnight.

Day 5:

ASHING (cont’d)

  1. Remove crucibles from furnace, using crucible tongs, and cool in dessicator for 2 hours. Turn off muffle furnace.
  2. Weigh bags and crucibles and record weight.
  3. Enter weights in spreadsheet.

DEFINITIONS

% NPE = nonpolar extracts such as fats, oils and waxes and soluble cell contents (carbs, lipids, pectin, starch, and soluble proteins) (dissolved in NDF wash)

% WS = hemi-cellulose and bound proteins (washed off in ADF wash)

%AS = cellulose (aka ligno-cellulose) (washed off in ADL wash)

% Lignin = lignin and some mineral ash

% Ash = minerals or non-organic material

% NDF = crude fiber

% ADF = complex of lignin and cellulose

The following fractions should add up to 100%:

%NPE + %WS + %AS + % Lignin + % Ash = 100%

OR

%NPE + % WS + % ADF = 100%

CALCULATIONS

Lignin Spreadsheet
Ash Weight = / ash & xbl wt - xbl wt
Ash corrected wt = / ash wt - ash wt of blank bag
Sample @ 105C = / sample wt - ((sample wt x (%H2O/100))
%NPE = / sample @ 105 - (NDF dry wt - Bag wt) x 100
sample @ 105
%WS = / (NDF dry wt - ADF dry wt) x 100
sample @ 105
%AS = / (ADF Dry wt - Lignin dry wt) x 100
sample @ 105
%Lignin = / (Lignin dry wt - bag wt - corrected ash wt) x 100
sample @ 105
%ash = / corrected ash wt x 100
sample @ 105
%NDF = / (NDF dry wt - Bag wt) x 100
sample @ 105
%ADF = / (ADF dry wt - Bag wt) x 100
sample @ 105
Dry wieght Spreadsheet
% H2O = / sample wt - (xbl & dry wt - xbl wt) x 100
sample wt
% Ash = / (xbl & ash wt - xbl wt) x 100
sample wt
Weigh out 0.45-0.55g of sample
Sample ID / Bag # / Bag Wt / Sample Wt / NDF Dry Wt / ADF Dry Wt / Lig Dry Wt / Xbl # / Xbl Wt / Ash Wt +Xbl / Ash Wt / Notes

Literature Cited

Ankom website

Ryan MG, Melillo JM and Ricca A. 1990. A comparison of methods for determining proximate carbon fractions of forest litter. Can. J. For. Res. 20:166-171.

Ashing Protocol

Day 1:

 Rinse crucibles with DI water.

 Shake off excess water and allow to air dry for ~ 12 minutes.

 Oven dry crucibles overnight at 105 C.

Day 2:

 Cool crucibles in dessicator for 1.5 hours.

NOTE: DO NOT TOUCH CRUCIBLES WITH BARE HANDS ONCE THEY HAVE BEEN CLEANED AND DRIED. WEAR GLOVES AND USE CRUCIBLE TONGS.

 Weigh crucible.

 Tare crucible weight and weigh out 0.1-0.5 grams of ground sample.

 Oven dry crucibles and samples overnight at 105 C.

 Turn on Muffle furnace and set to 150 C. Make sure the hood vent in rm 418 is also turned on.

Day 3:

 Cool samples in dessicator for 1.5 hours.

 Weigh samples.

 Place samples into the muffle furnace at 500 C for 5.5 hours.

 After 5.5 hours turn down temperature to 150 C and leave samples in the furnace overnight.

Day 4:

 Turn off muffle furnace and remove samples using crucible tongs.

 Cool samples in dessicator for 2 hours.

 Weigh samples and crucibles.

1