I sincerely hope this will be of some use to you in your research. Good luck!

I guess, spectral methods are fast and suitable for comparison study, but not very accurate.

Chemical methods like Klason lignin determination with 72% sulfuric  acid is relatively easy to perform. Acetyl Bromide method is another possibility to analyze lignin content in plant tissues.

Schwanninger, M., Hinterstoisser, B. (2002) Klason lignin: modification to improve the precision of the standardized determination. Holzforschung 56(2):161-166.

Hatfield, R., Fukushima, R.S. (2005) Can lignin be accurately measured? Crop Sci. 45:832– 839.

Hatfield, R.D., Brei, K., Grabber, J.H. (1996) Revising the acetyl bromide assay to optimize lignin determinations in forage plants. U.S. Dairy Forage Research Center 1996 Research Summaries. Madison, WI. pp. 63 – 65.

Hatfield, R.D., Grabber, J., Ralph, J., Brei, K. (1999) Using the acetyl bromide assay to determine lignin concentrations in herbaceous plants: some cautionary notes. J. Agric. Food Chem. 47:628 – 632.

Good luck!

Please find attached a nice article describing everything you want to know and more. Good luck! 

You may use Klason method or any derived from the former one /e. g. ASTM T 13 m 54 method/. They are based on two step hydrolysis of polysaccharides in H2SO4. First step is 1h hydrolysis with 68 - 72 % H2SO4  at  20 oC. The  second represents hydrolysis of fragments of polysaccharides /after first step/ to monomers /4h boiling in 3 to 4 % H2SO4. Disadvantage of these methods is presence of small portion /1 to few %/ of lignin passing into the hydrolysate. The amount of solluble lignin can be determined via UV spectroscopy when  the absoptivity ceoff. of lignin is known.

Simpler and reproducible results provides so called "acetylbromide method"  based on dissolution of the sample in an acetylbromide/anhydrous CH3COOH mixture at elevated temperature. Reaction mixture is submitted to UV, and the content of lignin in the sample is calculated from absorbance.

Regardless the method of lignin determination, all samples have to be rid of interferring compounds /polyphenols, tannins, aminoacids, pigments, salts, fats, fatty acids etc./ prior to lignin determinations. In your case I would submit the samples to extraction with polar solvent /methanol or ethanol/ using Soxhlet apparatus /8 hours/,  and than the samples free of polar extractives would be extracted with benzene-ethanol mixture, hexane, toluene or acetone in the same apparatus.

At the same time you can determine extractives in the samples, and use the data for calculation of lignin contents in the samples before extractions. 

To determine lignin (insoluble and soluble in acid), we use the Laboratory Analytical Procedure (LAP) NREL/TP 510-42618 (Technical Reports of the National Renewable Energy Laboratory). It is attached. Best regards.

Thanks one and all for your valuable comments and supporting materials you had sent.

While going through several articles, i found that Gas chromatography mass spectroscopy is  also a reliable technique for lignin estimation, however the sample extraction is not much clear. Off-line pyrolysis, Soxlet extraction and also extraction by shaking sample ( in a polar solvent) overnight in a rotary shaker followed by centrifugation is suggested.

You can determine lignin in leafs or in composted leafes also indirectly as a difference betwen dry weight of extractives free sample and corresponding dry weight of holocellulose. This indirect determination may be slightly distorted due to smaller amounts of residual lignin in the preparation, amounting to 0 - a few %.

I would prefere isolation and determination of holocellulose according to the method of Wise.

Procedure: 2 g of extractives free material  is weighted into 250 ml Erlenmayer flask containing 80 ml of distilled water, to which 0.25 ml of ice CH3COOH is added. The flask is placed into to 80 oC heated water bath. When  the inner temperature is equal to bath temp. 0.75 g of NaClO2 is added and the openning of flask is "sealed" with a small 25 ml Erlenmayer flask placed upside-dovw . The oxidative delignification proceds in 4 steps, each lasting an hour. After each  step /excluding the last one/ the above mentioned chemicals, in the same order, are added. In the end, the contents are cooled with adition of sufficient amount of ice to temp. moderately over 0. The isolation should be performed in a digestorium due to toxic Cl2 and Cl- oxides formation. 

The suspension is separated via vacuum filtration of the spent solution via S-3 crucible. The holocellulose in the crucible is then washed with ethanol,  acetone and dried in a vacuum or in thermostat at 103 oC

If neccessary, the content of residual lignin in the preparation can be determined via UV, or aproximately as Klason lignin.

Notice: the mentioned differential method might be possibly more convenient for your analysed material /sound leafs - composted leafs/. 

I suggest acetyl bromide method, the simplest method.

See here: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0110000