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    Environmental & Plant Biology
    Mailing address: Porter 315

    Total Carbon and Nitrogen ? (Analyzer Set Up)

    By Jared L. DeForest

    1. Turn on computer and wait until it finishes loading.
    2. Remove black carousel from top of C/N analyzer.
    3. Turn on (green switch) C/N analyzer and allow 2 minutes for initialization.
      1. If initialization is successful, two carousel alignment pins will line up on top.
    4. Return black carousel to top of C/N analyzer. Make sure the two receiving holes are aligned.
    5. Load Vario EL software
      1. Note furnace temperature on lower gray status bar, if it doesn’t read ambient (e.g., 23°C) then there is no PC-C/N communication.
      2. Only one copy of the software can be open at one time.
    6. Fully open valves on gas tanks. Note tank pressure, it must be above 200 psi.
    7. Check flow on C/N analyzer:
      1. Left flow meter should read ~1.0 bar; O 2 : 2.5 bar (blue tank).
      2. Right flow meter should read ~3.0 bars; He: 29 psi (orange tank).
      3. Round pressure gauge should read 1.25 bars.
    8. Always perform a leak check if system was repacked or pressure gauge is < 1.0 bars.
      1. Lower the He pressure to 18 psi.
      2. Plug white hoses in back of C/N analyzer with fittings.
      3. In the software go to options, misc., leak check, to start auto leak check.
      4. If you pass the test with < 80%, then contact Dr. DeForest.
      5. Remember to reset He pressure to 29 psi.
    9. Within the software, go to task bar and select Options, Parameter, (no password, click ok), enter values for furnace temperatures and click ok.
      1. Furnace 1 = 950°C
      2. Furnace 2 = 500°C
    10. Confirm that ovens are warming:
      1. Lower right status bar should read “heating up.”
      2. Lower left status bar, furnace temperatures should be rising.
    11. System is operational when furnaces attain target temperatures for at least 1 hour. It will take ~ 2 hours after turning on the furnace. For best results, let it run overnight.

    Total Carbon and Nitrogen – (Sample entry & Standards) (H3)

    Carousel tray should be set up with:

    • 2 empty (blank) tins
    • 2 run-in samples (conditioner, 1-3 mg of Acetanilide)
    • 3 standards (See reference standard section.)

    Oxygen setting for soil and litter should be 1, wood should be 3, and blank is 2.

    Set up Vario-EL spreadsheet prior to run (double click on sample lines to access input). The following is an example for soil (2-5% carbon) on the large carousel:

    Sample

    Name

    Wt. (mg)

    O2

    1

    Blank

    1.0

    2

    2

    Blank

    1.0

    2

    3

    Run-in

    ~2.5

    1

    4

    Run-in

    ~1.5

    1

    5

    Atro

    ~1.0

    1

    6

    Atro

    ~0.6

    1

    7

    Atro

    ~2.0

    1

    8 - 23

    [Sample 1-15]

    [balance]

    1

    24

    Atro

    ~1.2

    1

    25-40

    [Samples 16-31]

    [balance]

    1

    41

    Atro

    ~0.8

    1

    42-57

    [Samples 31-46]

    [balance]

    1

    58

    Atro

    ~2.5

    1

    59-74

    [Samples 47-62]

    [balance]

    1

    75

    Atro

    ~0.7

    1

    76-80

    [Samples 62-66]

    [balance]

    1

    If the analyzer has been inactive for several months, it is ok to start a carousel with a run-in to purge the system.

    Reference standard (Font color will be blue.)

    (Atropine – 70.56% C & 4.84% N) – Name must be ‘Atro’: Best for soil/litter/wood
    or
    (Acetanilide - 71.09% C & 10.36% N) – Name must be ‘Ace’: Best for biomass

    For most samples (atropine standard):

    SOIL—Weigh out the standards between 0.6 mg and 3.0 mg.

    • Half of the standards should be between 0.6 mg and 1.25 mg

    LITTER—Weigh out the standards between 1.25 mg and 4.5 mg.

    • Half of the standards should be between 1.75 mg and 3.00 mg

    Make sure each standard weight is at least 0.2 mg apart from the other standard weights.

    Refer to the table on the side of the fume hood for details.

    Total Carbon and Nitrogen (Sample Prep)

    Procedure – Pulverize soils

    Use the 8000M ball mill (Room 415); ask Dr. DeForest for permission before using.

    1. Add enough air-dry, sieved soil to fill the chamber half way (~15 g).
    2. Add several pulverizing balls.
    3. Pulverize for 1 min.
    4. Place pulverized soil in a labeled small vial.
    5. Store in a desiccator away from light.

    Procedure – Encapsulating samples

    Do not touch the tin with your hands, use forceps .

    1. Place a blank tin on the microbalance
    2. Tare balance so it reads 0.000.
    3. Remove tin and place within a 96-well plate.
    4. Add pulverized soil to tin using a micro scoop or spatula.
      • Do not add soil or litter to the tin while on the balance!
    5. Place back on scale.
      • If weight is off target, add or remove soil and re-weigh.
        Encapsulated weight for typical forest soils is between 25 and 30 mg.
        Organic soils/compost: 10-15 mg
        Subsoil/mineland: 45-50 mg
        Encapsulated weight for litter is between 3 and 4 mg.
      • If weight is on target, carefully fold tin over and form a plug with the press.
        It is very important that the encapsulated sample form a ball or thick disk.
    6. Place encapsulated sample back on scale.
    7. Press the yellow button on the microbalance to record weight in Vario EL software. (Optional)
    8. Always record weight of the soil plug in your lab notebook.
    9. Place sample in a 96-well plate, record position (e.g., A1, A2, A3…..H10, H11, H12). Store the 96- well plate in a desiccator until analysis.

    Total Carbon and Nitrogen – (Analyzing Samples)

    YOU MUST FOLLOW THESE METHODS FOR PUBLISHABLE RESULTS.

    REMEMBER TO ALWAYS BE PATIENT AND TAKE YOUR TIME.

    FALIURE TO CALIBRATE THE ANALYZER WILL YIELD INACCURATE RESULTS!

    • C/N analyzer should be at operating temperature for at least three (3) hours before running blanks! You should wait overnight if you repacked the reduction tube. Best method: let the system sit overnight to fully purge and condition the analyzer after maintenance.This should make it easier to calibrate the C/N analyzer.
    • To initiate run sequence, click on Auto Start button on command line.
    • DO NOT LEAVE THE C/N ANALYZER UNTIL THE END OF THE FIRST SAMPLE!This will take at least 70 minutes. Load your samples on the carousel after the blanks, run-ins, and two of the standards.
    • Blanks are used to confirm the system is working well. Second blank should stabilize carbon and nitrogen peaks (< 50). DO NOT CONTINUE TO RUN-INS UNLESS BLANK PEAKS ARE BELOW 50! Repeat blanks until peaks are below 50 or purge the system with a run-in and run another blank.
    • Run-ins are used to condition machine for the following standards. Run-ins are not part of the standard curve. Values should be consistent with little variation (i.e., < ± 0.3% for C & N). DO NOT CONTINUE TO STANDARDS UNLESS RUN-INS ARE SIMILAR.Repeat run-ins until % C & N are similar.
    • It is very important to have the correct sample weight. See sample prep page for details.
    • Each sample requires ~8-10 minutes, so it is around 12 hours for the large carousel (80 slots).
    • ALWAYS FILL OUT THE MAINTENANCE LOG ON THE C/N ANALYZER! See maintenance page for details.
    • Always record the data (% C and % N) in your lab notebook ASAP after the run. Save the run with either your name or the project followed by the date. (e.g. PAX_092709).

    Total Carbon and Nitrogen – (Maintenance)

    Please record the number of samples in your analyzing session on the maintenance log. The log is attached to the C/N analyzer. You must record the following:

    • Date
    • Full name and project
    • Type of sample (e.g., soil, leaves, litter)
    • The number of runs (this includes blanks, run-ins, standards, and samples) The pressure in psi of the He tank and the start and end of a session
    • The score (%) on the leak check test
    • Put a check mark if the ash trap, H 2 O absorption tube, combustion (oxidation) or reduction tubes were changed after your session.

    Contact Dr. DeForest if maintenance is required. Only a fully trained person may independently service the analyzer.

    Maintenance will be required shortly if maintenance display at bottom left corner is blinking.

    • Ash trap
      • Every 80 soil samples if tin wt. is < 50 mg
      • Every 60 soil samples if tin wt. is between 50 - 60 mg
      • Every 50 soil samples if tin wt. is between 60 - 80 mg
    • H 2 O – Absorption-U-Tube (i.e., H 2 O scrubber)
      • As needed (~200 soil sample or 150 litter samples)
      • Fully spent Sicapent is blue, partially spent Sicapent is green
    • Reduction tube = every 500 runs or until only 50 cm of the Cu is still copper in color
    • Combustion tube = every large carousel (~80 samples)

    Shut down

    • Within the software, go to task bar and select Options, Parameter, (no password, click ok), enter values for furnace temperatures and click ok.
      • Furnace 1 = 0°C
      • Furnace 2 = 0°C
    • Shut off gases at the tank. Turn valve clock-wise to close.
    • Let stand overnight
      • Do not turn off analyzer until furnaces are below 100°C.
    • Turn off (green switch) C/N analyzer
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