Using the Acetyl Bromide Assay To Determine Lignin Concentrations in Herbaceous Plants: Some Cautionary Notes

The acetyl bromide assay was developed to provide a rapid and sensitive method for quantifying lignin in woody plant species. The original procedure cautioned against prolonged reaction times and advised keeping the reaction temperature at 70 °C to prevent excessive carbohydrate degradation that would skew the absorption spectra. Characterization of the reaction conditions revealed that the acetyl bromide reagent readily degrades xylans, a prominent polysaccharide group within all lignified plants. This degradation results in increased absorbance in the 270−280 nm region that is used to quantify lignin. The degradation of xylans is temperature dependent and is exacerbated by the addition of perchloric acid. Lowering the reaction temperature to 50 °C and increasing the reaction time from 2 to 4 h allows complete lignin solubilization but minimizes degradation of the xylans. Keywords: Acetyl bromide lignin assay; lignin; cell wall; xylans; alfalfa; corn