The reaction of thioglycolic acid (4.7) with the benzyl alcohol groups in lignin (4.8) under acidic conditions and at elevated temperatures results in the formation of thioethers that render the derivatized lignin (4.9) soluble in an alkaline solution (Figure 4.2).
Figure 4-2. Reaction of thioglycolic acid with lignin. Adapted from Hatfield and Fukushima (2005).
The method was first described by Browning (1967) for 40-gram samples and then adapted for smaller samples (15 mg) by Bruce and West (1989). In this modified protocol, which is the most practical, the sample is incubated for 4 h. at 95°C in 1 mL 2 M HCl and 0.2 mL thioglycolic acid. The insoluble residue containing the lignin is recovered by centrifugation, washed three times in water, and the pellet is dissolved in 0.5 M NaOH. Non-lignin materials will not dissolve and are removed by centrifugation. The supernatant containing the thioglycolate lignin is then recovered by precipitation after addition of HCl and a 4-h. incubation at 4°C. The thioglycolate lignin is again recovered by centrifugation, dried, and dissolved in 0.5 M NaOH. An aliquot is diluted 40 times in 0.5 M NaOH and the absorbance at 280 nm is read. An in-vitro generated dehydrogenation lignin polymer (DHP) is used as a standard. The DHP is made by polymerizing coniferyl alcohol in the presence of hydrogen peroxide and horseradish peroxidase.
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).
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