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Ankom Fiber Analyzer Protocol

Author

Hobbie Lab

Goal

This protocol serves as a guide to use the Ankom Fiber Analyzer.

Important Note

At least 0.5 grams of sample is required for analysis.

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), 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. All calculations can be found in the Template spreadsheet in the protocols folder in the Cavender-Bares Lab University shared drive.

Supplies

All Ankom solutions can be ordered from Ankom:

Ankom Technology
140 Turk Hill Park
Fairport, NY 14450
phone: 716-425-3940
fax: 716-425-3941
www.ankom.com

Ankom Supplies Costs -> Updated 2/28/2019

  • Neutral Detergent solution - catalog # FND20 - 20L for $113
  • Acid Detergent 5x solution - catalog # FAD20 - 1 L (dilutes to 5 L)for $80, 20L for $93
  • 72% Sulfuric acid - catalog # FSA72 - 1 L for $34
  • Filter bags - catalog # F57 – 200 for $252
  • Ankom chamber (in lab 540 - Sarah Hobbie’s Lab)
  • Drying Oven
  • Scale
  • Tea Kettle
  • Timer
  • Nanopure water
  • 1L beaker
  • 2L beaker

Template

Link to template

Steps for sample preparation

  1. Grind samples using a 1 mm screen and dry.

  2. Label bags with special Ankom marker or industral permanent Sharpie. Label with a unique ID e.g. 1-24.

  3. Tare Ankom bag and record weight.

  4. Weigh out .5 g (± 0.05 g) of sample and record weight. Include a blank bag in each run (each batch of 24) for a blank bag correction.

  5. Seal the bag closed within 0.5 cm from the open edge using the heat sealer. Be sure to seal the blank bag too.

  6. Spread sample uniformly inside the filter bag by shaking and lightly flicking the bag to eliminate clumping.

  7. A maximum of 24 bags may be place in the bag suspender. 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

  1. 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.

  2. Place bag suspender into the chamber. 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.

  3. Just before the 75 minutes is up, heat 1800 ml nanopure water to boiling in the electric tea kettle.

  4. After 75 minutes turn Agitate and Heat OFF, open the drain valve and exhaust the hot solution BEFORE opening the lid. Be aware, the solution will be around 100° C. The neutral detergent can be dumped down the drain. WARNING: 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.

  5. After the solution has been exhausted, close the valve and open the lid. Pour in the 1800 ml of boiling water and just lower the lid but do not seal it. Agitate for 3-5 minutes, but do NOT Heat. Exhaust the rinse water down the sink. During the first rinse, heat 1800 ml of water for a second rinse. Rinse two more times for a total of 3 rinses.

  6. Remove the filter bags and gently press out the excess water. If detergent is still in the filter bags, please rinse again until the detergent is completely removed. Place them in the drying oven and dry completely. Dry them overnight at 65° C.

  7. Cool bags to room temperature and weigh all the bags. Record weights.

  8. Move on to ADF extraction.

NDF Calculations

\[blank\;bag\;correction\;(C_{NDF}) = {blank\;bag\;wt.\;after\;extraction \over blank\;bag\;tare\;wt.}\]
\[\begin{align} Percent\;washed\;off\; & = soluble\;cell\;contents \\ & = {((\;sample\;bag\;wt. + sample\;wt.) - bag\;wt.\;after\;NDF) * 100 \over sample\;wt.} \\ \end{align}\]
\[\begin{align} Percent\;left\;after\;NDF\; & = hemicellulose,\;bound\;proteins,\;cellulose,\;lignin,\;recalcitrants \\ & = {((bag\;wt.\;after\;NDF - (sample\;bag\;wt. * C_{NDF})) * 100) \over sample\;wt.} \\ \end{align}\]

ADF Extraction

  1. Dilute the 5x ADF solution to the desired volume for a run. Pour it into the chamber. Make sure the valve on the left side of the machine is closed!

    1900 ml 1x ADF = 380 ml 5x ADF solution + 1520 ml nanopure water
    1500 ml 1x ADF = 300 ml 5x ADF solution + 1200 ml nanopure water

  2. Place the bags in the bag suspender and place it in the chamber.

  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. Just before the 60 minutes is up, heat 1800 ml nanopure water to boiling in the electric tea kettle.

  5. After 60 minutes turn Agitate and Heat OFF, open the drain valve and exhaust the hot solution BEFORE opening the lid. Be aware, the solution will be around 100° C. The acid detergent solution should be exhausted into a waste container. (The waste can later be picked up by DEHS). WARNING: 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 the solution has been exhausted, close the valve and open the lid. Pour in the 1800 ml of boiling water and just lower the lid but do now seal it. Agitate for 3-5 minutes, but do NOT Heat. Exhaust the rinse water down the sink. During the first rinse, heat 1800 ml of water for a second rinse. Rinse two more times for a total of 3 rinses.

  7. Remove the filter bags and gently press out the excess water. Place them in the drying oven and dry completely. Dry them overnight at 65° C.

  8. Cool bags to room temperature in a desiccator and weigh all the bags. Record weights.

  9. Move on to ADL extraction.

ADF Calculations

\[blank\;bag\;correction\;(C_{ADF}) = {blank\;bag\;wt.\;after\;extraction \over blank\;bag\;tare\;wt.}\]
\[\begin{align} Percent\;washed\;off\; & = hemicellulose\;and\;bound\;proteins \\ & = {(((bag\;wt\;after\;NDF - (tare\;wt\;sample\;bag * C_{NDF})) - (bag\;wt\;after\;ADF - (tare\;wt.\;sample\;bag * C_{ADF})) * 100) \over sample\;wt.} \\ \end{align}\]
\[\begin{align} Percent\;left\;after\;ADF\; & = cellulose, lignin, and recalcitrants \\ & = {((bag\;wt.\;after\;ADF - (tare\;wt.\;sample\;bag * C_{ADF})) * 100) \over sample wt.} \end{align}\]

ADL Extraction

  1. Place dry bags in a 2 L beaker and cover with 72% sulfuric acid (~300 ml to cover 48 bags). 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.

  2. Place a 1 L beaker inside the 2 L beaker to keep bags submerged.

  3. Agitate bags at start and at 30-minute intervals for 3 hours. Do this by lifting the 1 L beaker up and down ~ 30 times.

  4. Just before the 3 hours is up, heat 1000 ml nanopure water.

  5. After 3 hours, pour off the sulfuric acid into a waste container (the waste can later be picked up by DEHS) and rinse with the boiling water. Rinse with boiling water until the pH is neutral. This takes about 6-7 rinses. Dump the rinses down the drain.

  6. Gently press out the excess water. Place them in the drying oven and dry completely. Dry them overnight at 65° C.

  7. Cool bags to room temperature in a desiccator and weigh all the bags. Record weights. At this time, you can ash your bags if you are concerned about sand, silica, etc. content.

ADL Calculations

\[blank\;bag\;correction\;(C_{ADL}) = {blank\;bag\;wt.\;after\;extraction \over blank\;bag\;tare\;wt.}\]
\[\begin{align} Percent\;washed\;off\; & = cellulose \\ & = {(((bag\;wt.\;after\;ADF - (tare\;wt.\;sample\;bag * C_{ADF})) - (bag\;wt.\;after\;ADL - (tare\;wt.\;sample\;bag * C_{ADL})) * 100) \over sample\;wt.} \\ \end{align}\]
\[\begin{align} Percent\;left\;after\;ADL\; & = lignin\;and\; recalcitrants \\ & = {((bag\;wt.\;after\;ADL - (tare\;wt.\;sample\;bag * C_{ADL})) * 100) \over sample\;wt.} \\ \end{align}\]

Ashing

  1. Tare crucible.

  2. Carefully remove sample from filter bag into crucible. Weigh sample and record weight.

  3. The crucibles cannot be labeled with tape as it will melt and sharpie will be burned off in the muffle furnace. So you must have good documentation of the placement of the crucibles with sample in the furnace.

  4. Set muffle furnace to 550° C and let samples ash for 3 hours.

    1. The remaining sample will be inorganic matter.

  5. Open door half-way and let cool for at least 30 minutes before removing.

  6. Use large tongs to remove glassware from furnace and continue to cool to room temperature.

Note: The ANKOM filter bags are able to be ashed in the muffle furnace; however, we have been separating the sample from the filter bags because it is less messy and smoky without the filter bags.

Last update: October 20, 2022