Oct 17, 2022
Measuring leaf carbon fractions with the ANKOM2000 Fiber Analyzer
- 1Université de Montréal
External link: www.caboscience.org
Protocol Citation: Jocelyne Ayotte, Etienne Laliberté 2022. Measuring leaf carbon fractions with the ANKOM2000 Fiber Analyzer. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9dyeqg3e/v1
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 17, 2022
Last Modified: October 17, 2022
Protocol Integer ID: 71446
Abstract
Here we describe the standardized protocol used by the Canadian Airborne Biodiversity Observatory (CABO) to measure carbon fractions in leaf samples, using the ANKOM2000 Fiber Analyzer. 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 manufacturer's protocols.
Materials
MATERIALS
Fiber Filter Bags F57VWR international LtdCatalog #CA-11022-996
Neutral detergent solutionVWR international LtdCatalog #CA11023-022
Acid detergent solutionVWR international LtdCatalog #CA11023-012
Sulfuric acidThermo Fisher ScientificCatalog #A300S-212
Sodium sulfiteThermo Fisher ScientificCatalog #S430-500
Alpha-amylaseVWR international LtdCatalog #CA11023-004
STEP MATERIALS
Alpha-amylaseVWR international LtdCatalog #CA11023-004
Sodium sulfiteThermo Fisher ScientificCatalog #S430-500
Alpha-amylaseVWR international LtdCatalog #CA11023-004
Paper pH StripsThermo Fisher ScientificCatalog #13-640-508
Protocol materials
Paper pH StripsThermo Fisher ScientificCatalog #13-640-508
Materials, Step 55
Fiber Filter Bags F57VWR InternationalCatalog #CA-11022-996
Materials
Neutral detergent solutionVWR InternationalCatalog #CA11023-022
Materials
Acid detergent solutionVWR InternationalCatalog #CA11023-012
Materials
Sulfuric acidThermo Fisher ScientificCatalog #A300S-212
Materials
Sodium sulfiteThermo Fisher ScientificCatalog #S430-500
In Materials, Materials, Step 16
Alpha-amylaseVWR InternationalCatalog #CA11023-004
In Materials, Materials, Materials and 2 steps
Before start
Leaf samples need the have been dried for 72 h at 65°C and then ground with a cyclone mill (2-mm screen).
Sample preparation
Sample preparation
Use a solvent resistant marker to label the filter bags to be used in the analysis.
Weigh and record the weight of each empty filter bag to the nearest 0.0001 g (in Fulcrum). Zero the balance.
Note
NOTE: Do not pre-dry filter bags. Any moisture will be accounted for by the blank bag correction.
Equipment
Sartorius Practum
NAME
Analytical balance
TYPE
Sartorius
BRAND
224-1S
SKU
LINK
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.
Include at least one empty bag in the run to determine the blank bag correction (C1).
Note
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.
Using a heat sealer, completely seal each filter bag closed within 4 mm of the top to encapsulate the sample.
Note
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.
Equipment
Impulse Heat Sealer
NAME
Heat Sealer
TYPE
Uline
BRAND
H-163
SKU
LINK
Spread the sample uniformly inside the filter bags by gently shaking the bags to eliminate clumping.
Neutral Detergent Fiber
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
NOTE: All nine trays must be used regardless of the number of bags being processed.
Verify that the hot water supply is on (room B-236).
Verify that the drain hose is securely positioned in an empty yellow waste bin.
Attach the Neutral Detergent (ND) solution hose to the Cubetainer and then to Port A on the instrument.
Add 50 ml of tap water in the alpha-amylase dispenser. Add 8.0 mL 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 20 g of Sodium sulfite and keep it for step 16.
Note
The ANKOM2000 will automatically add the amylase solution to the first and second rinse.
Alpha-amylaseVWR international LtdCatalog #CA11023-004
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.
Follow the instructions on the ANKOM2000 display:
Select NDF. (Wait to close the Vessel Lid.)
The screen will show: Insert samples. Press Enter.
Confirm hot water is on (>70 °C) and press START.
After the ND solution has been automatically inserted and agitation begins, manually add 20g of Na2SO3 and 4.0 ml of alpha-amylase.
20 g Na2SO3 4.0 mL alpha-amylase
Sodium sulfiteThermo Fisher ScientificCatalog #S430-500
Alpha-amylaseVWR international LtdCatalog #CA11023-004
Equipment
Fiber analyzer
NAME
Ankom
BRAND
A2000
SKU
LINK
Close the Vessel Lid.
Equipment
Fiber analyzer
NAME
Ankom
BRAND
A2000
SKU
LINK
This step takes about 01:30:00 .
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.
Safety information
HOT! Be careful!
Equipment
Fiber analyzer
NAME
Ankom
BRAND
A2000
SKU
LINK
Completely dry the filter bags in an oven at 102 ± 2°C for 3 h.
03:00:00
During that time, go to Step 20 and Flush Procedure (Step 21).
Equipment
Oven
NAME
Oven forced-air convection
TYPE
Fisher Isotemp
BRAND
15-103-0510
SKU
LINK
During that time, run a flush procedure if necessary.
Detach both cubitainers from ports A and B.
Note
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.
Note
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.
Flush procedure:
Verify that the drain hose is securely inserted into a waste bin.
Flush procedure:
Press the arrow keys on the Keypad until you see “Select Flush” on the Display.
Flush procedure:
Press ENTER on the Keypad.
Flush procedure:
Attach the Amylase Dispenser Assembly to port B.
Flush procedure:
Fill the dispenser with 100 ml of hot water.
Flush procedure:
Attach the Amylase Dispenser Assembly to port A.
Flush procedure:
Fill the dispenser with about 100 ml of hot water.
Note
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.
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 00:30:00 .
Note
NOTE: Do not use a conventional countertop or cabinet desiccator.
Weigh the filter bags to the nearest 0.0001 g and add record the weight (in Fulcrum).
Note
If not processing immediately with the Acid Detergent Fiber (ADF) steps, put the filter bags in the dessicator until the next step.
Remove the yellow waste bin and label it properly.
Note
Wait few hours to let the waste cool down before adding the label (it does not stick when it is hot).
Acid Detergent Fiber
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
NOTE: All nine trays must be used regardless of the number of bags being processed.
Insert the drain hose securely into an empty white waste bin.
Read the Temperature Controller on the right side of the instrument.
Note
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.
Attach the acid detergent (AD) solution hose to the Cubetainer and then to Port B on the instrument.
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.
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.
01:00:00
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.
Safety information
HOT! Be careful!
Completely dry the filter bags in an oven at 102 ± 2°C for 3 h.
03:00:00
Equipment
Oven
NAME
Oven forced-air convection
TYPE
Fisher Isotemp
BRAND
15-103-0510
SKU
LINK
During that time, remove the white waste bin and label it properly.
Note
The label will not stick if the content is hot. Wait a few hours to cool down before sticking the label.
During that time, run a flush procedure if necessary.
Detach both cubitainers from ports A and B.
Note
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.
Flush procedure:
Verify that the drain hose is securely inserted into a waste bin.
Flush procedure:
Press the arrow keys on the Keypad until you see “Select Flush” on the Display.
Flush procedure:
Press ENTER on the Keypad.
Flush procedure:
Attach the Amylase Dispenser Assembly to port B.
Flush procedure:
Fill the dispenser with 100 ml of hot water.
Flush procedure:
Attach the Amylase Dispenser Assembly to port A.
Flush procedure:
Fill the dispenser with 100 ml of hot water.
Note
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.
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
NOTE: Do not use a conventional desiccator container.
Weigh the filter bags to the nearest 0.0001 g and record the weight (in Fulcrum).
Equipment
Sartorius Practum
NAME
Analytical balance
TYPE
Sartorius
BRAND
224-1S
SKU
LINK
Note
If not proceeding immediately with the acid lignin method, place the filter bags in the dessicator until the next step.
Lignin method in beakers
Lignin method in beakers
Prepare a 72% H2SO4 solution or take a 250 ml bottle of 72% H2SO4.
Safety information
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.
Note
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.
.
Place dried filter bags with samples into a 2 L glass beaker and completely cover the bags with 72% H2SO4 (approximately 250 ml).
Note
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.
Place a 1 L beaker inside the 2 L beaker to keep the filter bags submerged.
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.
After 3 h, pour off the H2SO4 and rinse with tap water to remove all of the acid.
Note
If acid remains in the filter bags when they go into the drying oven, the samples will burn, leading to erroneous results.
Safety information
H2SO4 and rinse water must be poured in a white waste bin, with a pink label.
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.
Paper pH StripsThermo Fisher ScientificCatalog #13-640-508
Dry the bags in an oven at 100 °C for 3 h.
03:00:00
Equipment
Oven
NAME
Oven forced-air convection
TYPE
Fisher Isotemp
BRAND
15-103-0510
SKU
LINK
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
Ashing
Rinse 24 crucibles and covers with dH2O.
Dry the crucibles and covers in an oven at 150 °C for 1 h.
01:00:00
Equipment
Oven
NAME
Oven forced-air convection
TYPE
Fisher Isotemp
BRAND
15-103-0510
SKU
LINK
Cool down crucibles (with lids on) in a metal dessicator for 1h30min.
01:30:00
Equipment
Fisherbrand™ Stainless Steel Desiccator with Stainless Steel Shelves
NAME
Metal Dessicator
TYPE
Fisher
BRAND
S02123
SKU
LINK
Weigh empty crucibles, with lids, to the nearest 0.0001 g. Record the weight (in Fulcrum).
Note
Do not wear lab gloves when weighing in this section. Gloves cause static.
Equipment
Sartorius Practum
NAME
Analytical balance
TYPE
Sartorius
BRAND
224-1S
SKU
LINK
Fold filters bags into four and insert each bag into its crucible, with the corresponding sample number. Cover with the lid.
Put the crucibles (with lids on) in the muffle furnace at 500 °C for 5 h.
05:00:00
Equipment
Thermo Scientific™ Thermolyne™ Largest Tabletop Muffle Furnaces
NAME
Muffle furnace
TYPE
Thermolyne
BRAND
10-505-13
SKU
LINK
Change the temperature at 150 °C, O/N.
14:00:00
Equipment
Thermo Scientific™ Thermolyne™ Largest Tabletop Muffle Furnaces
NAME
Muffle furnace
TYPE
Thermolyne
BRAND
10-505-13
SKU
LINK
Turn off the muffle furnace and transfer the crucibles (with lids on) in a metal dessicator for 1h30min.
01:30:00
Equipment
Fisherbrand™ Stainless Steel Desiccator with Stainless Steel Shelves
NAME
Metal Dessicator
TYPE
Fisher
BRAND
S02123
SKU
LINK
Weigh crucibles with lids on to the nearest 0.0001 g. Record the weight (in Fulcrum).
Equipment
Sartorius Practum
NAME
Analytical balance
TYPE
Sartorius
BRAND
224-1S
SKU
LINK
Wash crucibles and lids with tap water.