Determination of lignin

Calculation. Subtract the weight of the empty crucible from that of the crucible plus ADF to obtain the weight of ADF. Divide by the sample weight and multiply by 100 to obtain the % ADF in the sample DM (air-dry matter for animal feeds). 

Procedure. Wear PPE - rubber gloves, face shield and lab coat when handling the permanganate-buffer solution. Place the weighed sintered glass crucibles containing the ADF residue one at a time into a stainless steel or polythene tray containing 2-3 cm cold water (a 400 x 320 x 50 mm tray will hold 48 

Notes on the reagents. The permanganate reagent oxidizes and dissolves the lignin and any tannins. The 2-methylpropan-2-ol enhances the wetting of the ADF fibres by the permanganate. The acetic acid in the buffer solution is to neutralize the alkali formed in the oxidation reaction  

Procedure. Ignite the dried crucible and residue from the lignin determination for 3 h in a furnace at 500°C. Allow to cool in a glass desiccator and weigh. 

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Determination of Lignin Content. Lignin content in plants (wood) is determined by direct or indirect methods (21). The direct method includes measurement of acid-insoluble (ie, Klason) lignin after digesting wood with 72% sulfuric acid to solubilize carbohydrates (22). The Klason lignin contents of representative lignifted materials are shown in Table 2. 

IR spectra were obtained on an FTIR spectrophotometer (Mattson Cygnus 100), using KBr discs containing 1% finely ground samples. For the method of determination of lignin remaining attached to/associated with XRPP fraction see our previous report (5). 

Van Soest, P.J. and Wine, R.H. (1968) Determination of lignin and cellulose in ADF with permanganate. Journal of the Association of Official Analytical Chemists 5M4), 780-785. 

It is unclear how accurate this method is for the determination of lignin content in grasses, since a fraction of the lignin from grasses appears to be readily soluble in acidic solutions, such as used in this procedure. This would result in underestimation of the actual lignin content (Hatfield and Fukushima, 2005). 

Fukushima, R. S., and Hatfield, R. D., 2004, Comparison of the acetylbromide spectrophotometric method with other analytical lignin methods for the determination of lignin concentration in forage samples, J. Agric. Food Chem. 52 3713-3720. 

Iiyama, K., and Wallis, A. F. A., 1990, Determination of lignin in herbaceous plants by an improved acetyl bromide procedure, J. Sci. Food Agric. 51 145-162. 

Johnson, D. B., Moore, W. E., and Zank, L. C., 1961, The spectro-photometric determination of lignin in small wood samples, Tappi 44 793-789. 

In considering the various methods proposed for the determination of lignin, it is important to bear in mind that none can be regarded as totally satisfactory. This situation arises from the fact that lignin has yet to be isolated in a pure or unmodified condition. As a result there is no definitive structural formula of lignin currently available upon which to base the development of a rational and quantitative measurement or to assess accurately the validity of the methods currently available. In addition, many of the methods which are commonly applied to the determination of lignin have fundamental defects which seriously undermine their accuracy. For these reasons, values reported in the literature may not always provide a reliable indication of the true lignin content of a material. 

In organizing the subject matter for this chapter, it was found expedient to divide the presentation into two main subsections, one dealing with the analytical methods applicable to lignin when initially present as a component of a solid mixture and the other focusing on the determination of lignin in solution. 

In contrast to the approach utilized in the direct determination of lignin, indirect methods do not involve the isolation of a lignin residue. Instead, the lignin content may be calculated as the difference between 100% and the polysaccharide content of an extractive-free sample or, more commonly, by measuring some characteristic structural functionality, property, or chemical response and relating the result to concentration. 

Kolboe and Ellefsen (1962) and Michell et al. (1965) provided preliminary results indicating the feasibility of employing infrared spectroscopy to determine the lignin content of finely ground wood and pulp samples embedded in potassium chloride. Further development and refinement of this technique have led to methods for determination of lignin based on multiple internal reflectance infrared spectrometry (Marton and Sparks 1967) and diffuse reflectance Fourier transform spectrometry (Schultz et al. 1985). Lignin contents have also estimated by 13C CP/MAS/NMR spectrometry (Haw et al. 1984, Hemmingson and Newman 1985) (see Chap. 4.5). 

The methods listed above have the advantage of being applicable to the determination of lignin in the solid state and, being nondestructive, they obviate the risk of concurrent chemical modification. In addition, these analyses required very small amounts of sample. 

A number of spectral methods for the determination of lignin consist essentially of totally dissolving the sample in a suitable solvent and measuring the UV-absorbance of the solution at a wavelength characteristic for lignin, usually 280 nm. Among the solvents used to dissolve the lignocellulosic material... 

Determination of Lignin in Wood and Pulp by the Acetyl Bromide Method... 

Another spectroscopic method for the determination of lignin in solution is that utilizing fluorescence spectrophotometry. This technique is reportedly capable of detecting lignin in concentrations two to three orders of magnitude... 

Alen R, Hartus T (1988) UV spectrophotometric determination of lignin from alkaline pulping liquors Cellul Chem Technol 22 613-618... 

Bagby MO, Cunningham RL, Maloney RL (1973) Ultraviolet spectral determination of lignin Tappi 56(4) 162-163... 

Bagby MO, Nelson GH, Helman EG, Clark TF (1971) Determination of lignin in non wood plant fiber sources Tappi 54 1876-1878... 

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