Lignin Method, Vitousek Lab, Stanford University

   This method follows that of Kenji Iiyama and Adrian Wallis; "Determination of Lignin in Herbaceous Plants by an Improved Acetyl Bromide Procedure"; J Sci Food Agric 1990; and R.D. Hatfield, J. Grabber, J. Ralph and K. Brei; "Using the Acetyl Bromide Assay To Determine Lignin Concentrations in Herbaceous Plants: Some Cautionary Notes", J. Agric. Food Chem. 1999.

Supplies and Reagents needed:

  • DI Water
  • Ethanol
  • Acetone
  • Ethyl ether
  • Acetic acid
  • Acetyl bromide
  • 2M Sodium hydroxide
  • 25% Acetyl bromide (v/v) in acetic acid
  • 24 20x150mm test tubes
  • Isotemp dry bath
  • Glass vacuum filter flask assembly
  • GF/A 2.4cm glass fiber filters
  • Filter forceps
  • 25ml glass graduated cylinder
  • 24 glass scintillation vials with teflon lined caps
  • 24 50ml volumetric flasks
  • Pipets to deliver 100µl, 2.5mland either 5 or 10ml
  • Glass funnel to fit volumetric flasks (60mm)
  • Vacuum pump  (light duty Barnant pump)



    1. To each test tube add 10 - 15 mg of dry ground sample. Samples are best if ground to a powder, but will work up to a maximum of a 20 mesh screen. Also, sample weights may range from 2 to 40mg and still give good results, but using at least 10mg insures more accurate mass, and using no greater than 15mg insures complete degestion. Always use one tube as a procedural blank and at least two tubes for "standards".

    2. To each tube add 10ml DI H2O. Place tubes in dry block at 65°C (set at 85 on our block); heat for one hour, stirring every 10 minutes.
    3. Filter sample through GF/A glass fiber filter and rinse three times with each of the following solutions in this order: water>ehanol>acetone>diethyl ether. Allow a couple minutes for each rinse.

    4. Place filter disk in glass 20ml scitillation vial (without lids) and heat overnight at 70°C.
    5. To each vial add 2.5ml of 25% AcBr in acetic acid.
    6. Place vials in 50°C oven for 2 hours with lids. Swirl occasionally.

    7. Cool samples in refrigerator. Prepare volumetric flasks with 10ml of 2N sodium hydroxide, and 12ml of acetic acid. Transfer each sample from scintillation vials to volumetric flasks. Rinse filter paper well with acetic acid into the volumetric and bring to volume with acetic acid.

    8. Allow samples to settle for at least an hour, but it is best to leave them overnight and measure them in the morning. Measure absorbance at 280nm.


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