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A new simple and safe method for quantifying lignin content in lignocellulosic biomass is described. The approach consists of measuring the absorbance of a solution of whole biomass dissolved in the ionic liquid 1-n-butyl-3-methyl imidazolium chloride, [Bmim][Cl], at 440 nm via ultraviolet- (UV-) visible spectrophotometry. An extinction coefficient for a lignin standard, highly pure lignin isolated from biomass through an organosolv process, is used in conjunction with the Beer- Lambert Law to calculate the lignin concentration. Principal component analysis (PCA) of Fourier Transform-Infrared (FTIR) spectra collected for several different lignin standards was performed to understand the differences in their chemical structure and composition (e.g., the relative amounts of syringyl and guaiacyl units). A rapid FTIR analysis of the whole biomass sample with unknown lignin content is required to assist in the proper selection of the lignin standard for the subsequent spectrophotometric analysis. The proposed method was tested and validated on two biomass types: Yellow poplar and Southern pine. The spectrophotometric approach yielded lignin contents for Yellow poplar and Southern pine of 25.7 ± 1.1% and 26.7 ± 0.7%, respectively, which are comparable to the values obtained by a standard wet chemical protocol, 25.1% ± 0.7 and 26.6 ± 0.4%, respectively. [ABSTRACT FROM AUTHOR]