Finely Pulverized Low-Moisture (FPL) Gel-State NMR for Near-Completely Solubilized Whole Plant Cell Walls

High-resolution NMR analysis of heterogeneous, insoluble lignocellulosic materials is a significant challenge because poor magnetic field homogeneity and restricted molecular mobility often hinder the acquisition of reproducible solution-state NMR spectra. In studies of lignocellulosic biomass and plant cell walls, gel-state NMR methods have enabled important advances. However, practical limitations in sample preparation, shimming stability, and spectral broadening have constrained method robustness and routine use. Here, we present a finely pulverized low-moisture (FPL) gel-state NMR method that enables the reliable acquisition of high-quality two-dimensional (2D) HSQC spectra from intact whole plant cell walls without chemical pretreatment. Systematic evaluation of drying protocols, ball-milling efficiency, and solvent composition shows that moisture control and particle fineness are the primary factors influencing gel homogeneity and magnetic field shimming performance. Use of a DMSO-d₆/DMF-d₇ (4:1, v/v) solvent system produces gels with reproducible chemical shifts, narrow linewidths, and well-resolved heteronuclear correlations. This stability allows standard automated shimming and acquisition protocols to be applied reliably. The method is validated across diverse types of biomass, establishing a robust NMR sample-preparation framework for whole plant cell wall analysis and extending the practical reach of solution-state NMR methodologies.