Quantification of Lignin and Its Structural Featuresin Plant Biomass Using 13C Lignin as Internal Standardfor Pyrolysis-GC-SIM-MS

摘要

Understanding the mechanisms underlying plant biomass recalcitrance at the molecular level can only be achieved by accurate analyses of both the content and structural features of the molecules involved. Current quantification of lignin is, however, majorly based on unspecific gravimetric analysis after sulfuric acid hydrolysis. Hence, our research aimed at specific lignin quantification with concurrent characterization of its structural features. Hereto, for the first time, a polymeric ¹³C lignin was used as internal standard (IS) for lignin quantification via analytical pyrolysis coupled to gas chromatography with mass-spectrometric detection in selected ion monitoring mode (py-GC-SIM-MS). In addition, relative response factors (RRFs) for the various pyrolysis products obtained were determined and applied. First, ¹²C and ¹³C lignin were isolated from nonlabeled and uniformly ¹³C labeled wheat straw, respectively, and characterized by heteronuclear single quantum coherence (HSQC), nuclear magnetic resonance (NMR), and py-GC/MS. The two lignin isolates were found to have identical structures. Second, ¹³C-IS based lignin quantification by py-GC-SIM-MS was validated in reconstituted biomass model systems with known contents of the ¹²C lignin analogue and wasshown to be extremely accurate (>99.9%, R² > 0.999)andprecise (RSD < 1.5%). Third, ¹³C-IS based lignin quantificationwas applied to four common poaceous biomass sources (wheat straw,barley straw, corn stover, and sugar cane bagasse), and lignin contentswere in good agreement with the total gravimetrically determined lignincontents. Our robust method proves to be a promising alternative forthe high-throughput quantification of lignin in milled biomass samplesdirectly and simultaneously provides a direct insight into the structuralfeatures of lignin.