TITLE: Measuring leaf carbon fractions with the ANKOM2000 Fiber Analyzer
AUTHORS: Jocelyne Ayotte, Etienne Laliberté

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Here we describe the standardized protocol used by the </span><a href="http://www.caboscience.org" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Canadian Airborne Biodiversity Observatory</span></a><span> (CABO) to measure carbon fractions in leaf samples, using the </span><a href="https://www.ankom.com/product-catalog/ankom-2000-automated-fiber-analyzer" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">ANKOM2000 Fiber Analyzer</span></a><span>. Prior to the analysis, leaf samples are oven-dried, and then ground with a cyclone mill (2-mm screen). Then, carbon fractions are measured using a sequential digestion procedure to measure neutral detergent fiber (NDF, acid detergent fiber (ADF), acid detergent lignin (ADL). Finally, the residues are ashed in a muffle furnace to determine inorganic recalcitrant materials. This protocol is based on the </span><a href="https://www.ankom.com/technical-support/fiber-analyzer-a2000" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">manufacturer's protocols</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Sample preparation]
Use a solvent resistant marker to label the filter bags to be used in the analysis.
?. [Sample preparation]
Weigh and record the weight of each empty filter bag to the nearest 0.0001 g (in Fulcrum). Zero the balance.
NOTE: Do not pre-dry filter bags. Any moisture will be accounted for by the blank bag correction.
?. [Sample preparation]
Place 0.45–0.50 g of prepared sample in up to 23 of the bags and record the weight to the nearest 0.0001 g (in Fulcrum) of each. Avoid placing the sample in the upper 4 mm of the bag.
?. [Sample preparation]
Include at least one empty bag in the run to determine the blank bag correction (C1).
NOTE: A running average blank bag correction factor (C1) should be used in the calculation of fiber. The inclusion of at least one blank bag in each run is mainly used as an indicator of particle loss. A C1 larger than 1.0000 indicates that sample particles were lost from filter bags and deposited on the blank bag during the extraction. Any fiber particle loss from the filter bags will generate erroneous results. If particle loss is observed then the grinding method needs to be evaluated.
?. [Sample preparation]
Using a heat sealer, completely seal each filter bag closed within 4 mm of the top to encapsulate the sample.
NOTE: Use sufficient heat to completely seal the filter bags and allow enough cool time (2 s) before removing each bag from the heat sealer.
?. [Sample preparation]
Spread the sample uniformly inside the filter bags by gently shaking the bags to eliminate clumping.
?. [Neutral Detergent Fiber]
Place up to 3 bags on each of eight Bag Suspender Trays (maximum of 24 bags). Stack the trays on the center post of the Bag Suspender with each level rotated 120 degrees in relation to the tray below it. Place the empty 9th tray on top.
NOTE: All nine trays must be used regardless of the number of bags being processed.
?. [Neutral Detergent Fiber]
Verify that the hot water supply is on (room B-236).
?. [Neutral Detergent Fiber]
Attach the Neutral Detergent (ND) solution hose to the Cubetainer and then to Port A on the instrument.
?. [Neutral Detergent Fiber]
Verify that the drain hose is securely positioned in an empty yellow waste bin.
?. [Neutral Detergent Fiber]
Open the Vessel Lid and insert the Bag Suspender with bags into the Vessel and place the Bag Suspender weight on top of the empty 9th tray to keep the Bag Suspender submerged.
?. [Neutral Detergent Fiber]
Follow the instructions on the ANKOM2000 display: Select NDF. (Wait to close the Vessel Lid.)
?. [Neutral Detergent Fiber]
Confirm hot water is on (>70 °C) and press START.
?. [Neutral Detergent Fiber]
After the ND solution has been automatically inserted and agitation begins, manually add 20g of Na2SO3 and 4.0 ml of alpha-amylase.
[Na2SO3]
[alpha-amylase]
?. [Neutral Detergent Fiber]
Close the Vessel Lid.This step takes about .
?. [Neutral Detergent Fiber]
When the NDF extraction and rinsing procedures are complete, open the Vessel Lid and remove the filter bags. Gently press out excess water from the bags.
HOT! Be careful!
?. [Neutral Detergent Fiber]
Completely dry the filter bags in an oven at 102 ± 2°C for 3 h. During that time, go to Step 20 and Flush Procedure (Step 21).
?. [Neutral Detergent Fiber]
When dry, remove the filter bags from the oven and immediately place them directly into a collapsible desiccant pouch and flatten it to remove any air. Cool to ambient temperature for .
NOTE: Do not use a conventional countertop or cabinet desiccator.
?. [Neutral Detergent Fiber]
Weigh the filter bags to the nearest 0.0001 g and add record the weight (in Fulcrum).
If not processing immediately with the Acid Detergent Fiber (ADF) steps, put the filter bags in the dessicator until the next step.
?. [Neutral Detergent Fiber]
Remove the yellow waste bin and label it properly.
Wait few hours to let the waste cool down before adding the label (it does not stick when it is hot).
?. [Acid Detergent Fiber]
Place up to 3 bags on each of eight Bag Suspender Trays (maximum of 24 bags). Stack the trays on the center post of the Bag Suspender with each level rotated 120 degrees in relation to the tray below it. Place the empty 9th tray on top.
NOTE: All nine trays must be used regardless of the number of bags being processed.
?. [Acid Detergent Fiber]
Insert the drain hose securely into an empty white waste bin.
?. [Acid Detergent Fiber]
Read the Temperature Controller on the right side of the instrument.
If the temperature is higher than 20 °C, cool the Vessel as follows: a. Fill the Vessel with cold water. b. When the Temperature Controller reads 20 °C, run the Flush Procedure to drain the water. c. Repeat steps a and b if necessary.
?. [Neutral Detergent Fiber]
During that time, run a flush procedure if necessary. Detach both cubitainers from ports A and B.
The Flush procedure allows you to clean the system with water. This procedure should be used when changing from one procedure to another (e.g., when preparing to do an ADF analysis after completing an NDF analysis) or when doing an NDF procedure after a previous NDF procedure with more than 2 h between procedures, or before storing the instrument.
Amylase tends to be sticky. You should always clean the Amylase Dispenser Assembly and flush Port B after every NDF procedure unless the next procedure you are running is another NDF, and you run it within 2 h of the previous procedure.
?. [Acid Detergent Fiber]
Attach the acid detergent (AD) solution hose to the Cubetainer and then to Port B on the instrument.
?. [Acid Detergent Fiber]
Open the Vessel Lid and insert the Bag Suspender with bags into the Vessel and place the Bag Suspender Weight on top of the empty 9th tray to keep the Bag Suspender submerged.
?. [Acid Detergent Fiber]
Follow the instructions on the ANKOM2000 display: a. Select ADF. b. Close the Vessel Lid. c. Confirm hot water is on (>70°C). d. Press START.
?. [Acid Detergent Fiber]
When the ADF extraction and rinsing procedures are complete, open the Vessel Lid and remove the filter bags. Gently press out excess water from the bags.
HOT! Be careful!
?. [Acid Detergent Fiber]
Completely dry the filter bags in an oven at 102 ± 2°C for 3 h.
?. [Acid Detergent Fiber]
During that time, run a flush procedure if necessary. Detach both cubitainers from ports A and B.
The Flush procedure allows you to clean the system with water. This procedure should be used when changing from one procedure to another (e.g., when preparing to do an ADF analysis after completing an NDF analysis) or when doing an NDF procedure after a previous NDF procedure with more than 2 h between procedures, or before storing the instrument.
?. [Acid Detergent Fiber]
Remove the filter bags from the oven, immediately place them into a collapsible desiccant pouch, and flatten it to remove any air. Cool to ambient temperature.
NOTE: Do not use a conventional desiccator container.
?. [Acid Detergent Fiber]
Weigh the filter bags to the nearest 0.0001 g and record the weight (in Fulcrum).
If not proceeding immediately with the acid lignin method, place the filter bags in the dessicator until the next step.
?. [Acid Detergent Fiber]
During that time, remove the white waste bin and label it properly.
The label will not stick if the content is hot. Wait a few hours to cool down before sticking the label.
?. [Lignin method in beakers]
Prepare a 72% H2SO4 solution or take a 250 ml bottle of 72% H2SO4. .
Danger, highly corrosive.Exothermic reaction when mixing H2SO4 and water. ALWAYS ADD ACID TO WATER (slowly) AND NOT THE OPPOSITE!Wear gloves, labcoat, safety glasses.Work under the chemical hood.
Under the chemical hood, place a stirring plate,  2 L glass beaker and a magnetic stir bar. Use a cylinder to measure 242 ml of dH2O and pour in the beaker. Start stirring (slow speed). Use a glass cylinder to measure 758 mL of H2SO4 and SLOWLY pour into the beaker. Wait at least 1 h to for the solution to cool down.In a 1 L glass cylinder, adjust the final volume to 1 L with dH2O. When completely cooled, aliquot in 250 ml bottles.
?. [Lignin method in beakers]
Place dried filter bags with samples into a 2 L glass beaker and completely cover the bags with 72% H2SO4 (approximately 250 ml).
Filter bags must be completely dry and at ambient temperature before adding concentrated acid. If moisture (even ambient moisture) is present in the bags, heat generated by the H2SO4 and H2O reaction will adversely affect the results.
?. [Lignin method in beakers]
Place a 1 L beaker inside the 2 L beaker to keep the filter bags submerged.
?. [Lignin method in beakers]
After 3 h, pour off the H2SO4 and rinse with tap water to remove all of the acid.
If acid remains in the filter bags when they go into the drying oven, the samples will burn, leading to erroneous results.
H2SO4 and rinse water must be poured in a white waste bin, with a pink label.
?. [Lignin method in beakers]
Repeat rinses until the pH paper shows neutral color when touching the bags. Handle the bags gently during rinsing. Fine lignin particles can exit the filter bag if not handled carefully.
?. [Lignin method in beakers]
Dry the bags in an oven at 100 °C for 3 h.
?. [Lignin method in beakers]
Remove the filter bags from the oven and place them directly into a Desiccant/Moisture Stop pouch. Flatten the pouch to remove air. Cool to ambient temperature and weigh the bags to the nearest 0.0001 g. Record the weight (in Fulcrum).
?. [Ashing]
Rinse 24 crucibles and covers with dH2O.
?. [Ashing]
Dry the crucibles and covers in an oven at 150 °C for 1 h.
?. [Ashing]
Cool down crucibles (with lids on) in a metal dessicator for 1h30min.
?. [Ashing]
Weigh empty crucibles, with lids, to the nearest 0.0001 g. Record the weight (in Fulcrum).
Do not wear lab gloves when weighing in this section. Gloves cause static.
?. [Ashing]
Fold filters bags into four and insert each bag into its crucible, with the corresponding sample number. Cover with the lid.
?. [Ashing]
Put the crucibles (with lids on) in the muffle furnace at 500 °C for 5 h.
?. [Ashing]
Change the temperature at 150 °C, O/N.
?. [Ashing]
Turn off the muffle furnace and transfer the crucibles (with lids on) in a metal dessicator for 1h30min.
?. [Ashing]
Weigh crucibles with lids on to the nearest 0.0001 g. Record the weight (in Fulcrum).
?. [Ashing]
Wash crucibles and lids with tap water.
?. [Acid Detergent Fiber]
Flush procedure:Verify that the drain hose is securely inserted into a waste bin.
?. [Acid Detergent Fiber]
Flush procedure: Press the arrow keys on the Keypad until you see “Select Flush” on the Display.
?. [Acid Detergent Fiber]
Flush procedure: Press ENTER on the Keypad.
?. [Acid Detergent Fiber]
Flush procedure:Attach the Amylase Dispenser Assembly to port B.
?. [Acid Detergent Fiber]
Flush procedure:Fill the dispenser with 100 ml of hot water.
?. [Acid Detergent Fiber]
Flush procedure:Attach the Amylase Dispenser Assembly to port A.
?. [Acid Detergent Fiber]
Flush procedure:Fill the dispenser with 100 ml of hot water.
If you press and hold the START key on the Keypad during the Flush operation, water will flow into the Vessel. This will rinse the bottom of the Vessel, but it will not rinse all the way to the top. If you need to rinse anything from the top part of the inside of the Vessel, pour hot water into the Vessel as needed during the Flush operation.
?. [Neutral Detergent Fiber]
Add 50 ml of tap water in the alpha-amylase dispenser. Add  of alpha-amylase and enough tap water to fill the dispenser (until the line). Attach the Amylase Dispenser Assembly to Port B on the instrument.Keep the pipette and alpha-amylase bottle next to the instrument for step 16. Weigh of Sodium sulfite and keep it for step 16.
8 ml
20 g
The ANKOM2000 will automatically add the amylase solution to the first and second rinse.
?. [Neutral Detergent Fiber]
The screen will show: Insert samples. Press Enter.
?. [Lignin method in beakers]
Agitate the bags at the start and at 30-minute intervals by gently pushing and lifting the 1 L beaker up and down approximately 30 times.
?. [Neutral Detergent Fiber]
Flush procedure:Verify that the drain hose is securely inserted into a waste bin.
?. [Neutral Detergent Fiber]
Flush procedure: Press the arrow keys on the Keypad until you see “Select Flush” on the Display.
?. [Neutral Detergent Fiber]
Flush procedure: Press ENTER on the Keypad.
?. [Neutral Detergent Fiber]
Flush procedure:Attach the Amylase Dispenser Assembly to port B.
?. [Neutral Detergent Fiber]
Flush procedure:Fill the dispenser with 100 ml of hot water.
?. [Neutral Detergent Fiber]
Flush procedure:Attach the Amylase Dispenser Assembly to port A.
?. [Neutral Detergent Fiber]
Flush procedure:Fill the dispenser with about 100 ml of hot water.
If you press and hold the START key on the Keypad during the Flush operation, water will flow into the Vessel. This will rinse the bottom of the Vessel, but it will not rinse all the way to the top. If you need to rinse anything from the top part of the inside of the Vessel, pour hot water into the Vessel as needed during the Flush operation.