A simple method to measure lignin

T1 What methods are available to estimate lignin?
T2 What are their shortcomings?
T3 An alternative method to determine lignin
T4 Calculations
T5 Further reading

     Topic 1: What methods are available to estimate lignin?
Lignin is a compound that is highly abundant in a range of plant species and heavily shapes the ecological niche of such plants. It can take up to 20% of the leaf biomass of woody species, and up to 30% in tree trunks. Lignin is a polymer of various constituents, most notably 3 phenylpropanoids. These are connected with a variety of cross-linkages, the reason why it is so difficult to decompose lignin.

Although ecologically highly relevant, it is not so easy to determine the concentration of lignin. Two methods have been applied frequently. First, plant material is treated with increasingly stronger and more aggressive chemicals, until a fraction remains that is even not dissolved in 72% H2SO4. This residue is determined gravimetrically and termed 'Klason' lignin. Alternatively, plant material can de digested with methylbromide, after which the building blocks, the three monolignols, are formed again (Morison 1972). Because of their aromatic nature, they absorb in the UV band, and by assuming that these building blocks represent a certain proportion of the total mass of lignin, an estimate of the lignin concentration in the original material can be made. For non-invasive methods, see Hatfield & Fukushima (2005).

     Topic 2: What are there shortcomings??
The first shortcoming of any lignin determination is that there is no pure lignin available that can serve as a standard for calibration. This is one uncertainty to live with. The Klason method works with aggressive chemicals, and probably measures a number of compounds. The method of Morison (1972) is probably more restrictive, but even more unpleasant to work with. If you have ever seen liquid methyl bromide slowly creeping out of a beaker and you know what it can do, you do not get very enthusiastic about that determination either. Moreover, you do not know to what extent the proportion of its three building blocks may vary between species or treatments.

     Topic 3: An alternative method to determine lignin
So it would be nice to have an alternative method, cleaner than both the Klason and Morison method. Such an alternative may be the following: if you extract plant material with chloroform/methanol, you remove all kind of soluble and lipophylic material such as sugars and lipids, etcetera. (See here for the overall extraction scheme). At the same time, a number of proteins will precipitate. The residue of this extraction (R2 in the extraction scheme) can be treated with 3% HCl. This will remove non-structural carbohydrates, such as starch and fructans that resisted the first extraction. The residue that is left (R3) is a mixture of cellulose, hemicellulose, cell wall protein, precipitated other proteins, and possibly some minerals like silica etcetera. If you ash part of this material, you know the mineral fraction, which is usually rather small, but extremely important to know. Deduct that from the total weight of R3 that is measuredOn the other part of R3 you can do a CN-analysis. Assuming that the N comes from protein, with an overall composition of 16% N and 53% C, you can estimate the amount of protein in the residue, and deduct that as well. What is left after the deduction is a mixture of cellulose/hemicelluloses on the one hand and lignin on the other. Knowing the mineral content and the [C] of protein, you can calculate what the [C] is of this mixture. Cellulose and hemi-cellulose have a very different C-concentration (circa 43%) than lignin (circa 71%). So, if you have calculated what the C-concentration of the mixture was, you can also determine its relative proportion (hemi-)cellulose and lignin, and in the end you can calculate back what the concentration must have been in the plant material you started with.
This method, described in Poorter & Villar (1997), except for the mineral correction that can be found in Aerts (????) relies on a number of assumption, and no doubt will not only specifically measure lignin, but probably also some other secondary compounds of 'nasty' nature, like the Klason method. It also cannot be calibrated, like any of the other methods. But contrary to the Klason and Morison method, it is clean, and more friendly for the environment and personal health.

     Topic 4: Calculations
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     Topic 5: Further reading
  • Hatfield R & Fukushima RS (2005) Can Lignin Be Accurately Measured? Crop Sci. 45: 832-839.
  • Morrison IM (1972) A semi-micro method for the determination of lignin and its use in predicting the digestibility of forage crops. J. Sci. Food Agric. 23, 455-463.
  • Morrison IM, Asiedu EA, Stuchbury T & Powell AA (1995) Determination of lignin and tannin contents of cowpea seed coats. Ann. Bot. 76: 287-290.
  • Poorter & Villar (1997) Chemical composition of plants: causes and consequences of variation in allocation of C to different plant constituents. In: Plant Resource Allocation. Bazzaz F & Grace J (eds). Academic Press, New York, pp. 39-72.