Determination of lignin in herbaceous plants by an improved acetyl bromide procedure

Abstract

A B S T R A C TA modified acetyl bromide procedure is proposed for the spectrophotometric determination of lignin in herbaceous plants. The (Browning 1967) to the analysis gives an overestimated value for the lignin content, as proteins co-precipitate with the lignin (Norman and Jenkins 1934). This has led to methods in which the proteins were removed, either by an enzymic pretreatment with pepsin (Crampton and Maynard 1938) or by an acidic detergent treatment (Van Soest 1963) before digestion with the 72% sulphuric acid. In addition, the cinnamic acids are probably rendered soluble in the acidic medium and are estimated as acid-soluble lignin (Lai and Sarkanen 1971), which in turn would lead to inflated values for the total lignin content.Johnson et a1 (1961) devised a method for determining lignin in wood which is rapid and requires only small samples. In this method, the woodmeal samples are dissolved in a solution of 25 % w/w acetyl bromide (AcBr) in acetic acid at 70"C, and the lignin contents are obtained by measurement of the ultraviolet (UV) absorbance of the resulting solutions at 280 nm. The method requires finely ground samples, which are sometimes not completely dissolved after the digestion. We recently proposed an improved AcBr procedure based on adding perchloric acid (HCIO,) to the digesting medium, which gave faster and complete dissolution of the samples (Iiyama and Wallis 1988a al(1964), to obtain reference lignin values. The AcBr method was found to be advantageous for both grasses and legumes in that proteins did not give rise to UV absorbance at 280 nm, although the chemically linked cinnamic acid moieties did give absorbance and were measured as lignin. In this report we describe the application of our improved AcBr method to a variety of herbaceous plant samples, and we propose the adoption of an alternative to Morrison's procedure for calculating lignin contents from the UV absorption of the AcBr digests at 280 nm. Use of the new calculation procedure gives lignin values for non-legume samples which are consistent with nitrobenzene oxidation data, but are significantly lower than those obtained by the application of Morrison's method (1 972a). EXPERIMENTAL InstrumentsUV spectra were measured on a Varian Superscan 3 spectrophotometer. Fourier transform infrared (FT-IR) spectra were obtained for KBr discs (1 % sample, 64 scans) on an Alpha Centauri FT-IR spectrometer (Mattson Instruments Inc, Madison, Wis, USA). Gas chromatography (GC) was carried out on a HewlettPackard 5830A instrument equipped for operation with capillary columns and flame ionisation detection. GC-mass spectrometry (GC-MS) was performed on a Hewlett-Packard 5995A instrument. Improved acetyl bromide method for lignin in herbaceous plants 141 Chemicals and materialsThe phenol 3-ethoxy-4-hydroxybenzaldehyde, the phenolic acids (E)-pcoumaric acid and (E)-ferulic acid, the proteins casein and trypsin, and the amino acid tyrosine, were obtained from commercial sources.The wheat (Triticum vulgare Vill) internode meal was f...

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