heteronuclear single quantum coherence (HSQC)) has significantly
increased spectral resolution and sensitivity [26]. Pretreatment
combined with cellulolytic enzyme lignin extraction [30] offers relatively
higher yield with respect to the mill wood lignin extraction
[31]. Unfortunately, lignin covalently attached to cellulosic moieties,
and enzyme adsorption onto lignin, poses additional challenges
for lignin isolation [32]. Recently, a new solution-state
2D-HSQC NMR spectroscopic method was reported to measure
the acetylated plant cell wall using a whole cell wall dissolving system.
Using this approach an analytical signal for lignin content
with the necessity of lignin isolation. As a result, homogeneity of
dissolved biomass in a solution system is required to visualize all
the signals, and deduce the 1JC-H dependence of these signals. In
practice, the reliability of the HSQC-based method for quantification
is compromised by a number of issues, including a wide range
in effective T2 relaxation times due to sample polydispersity, coupling
constant deviations, homonuclear couplings, and sample
dependent resonance offsets [33].While 2D-HSQC NMR represents
an innovative method ideal for investigating structural changes in
lignin [34], it still has limitations.
To circumvent exhaustive extraction methodology, and diminishing
results over time to determine insoluble lignin in solution,
this study develops a solid-state method for quantitative and direct
measurement of lignin in biomass. Quantitative solid-state NMR
was first reported in middle 1990s [35], but its application was
limited because of low sensitivity and resolution. With the development
of cross polarization and MAS technology along with the
greater availability of ultra-high field NMR magnets, the resolution
and sensitivity of 13C CP/MAS ssNMR has been greatly enhanced.
However, until now, 13C CP/MAS ssNMR has only been used for
qualitative analysis for the insoluble components of biomass, such
as cellulose [17] and lignin. An early report on structure changes in
lignin and plant polysaccharides after fungal degradation using
solid-state NMR first suggested the use of this method on lignocellulosic
biomass [36]. The chemical structure and heterogeneity of
two lignin structures from Loblolly Pine were subsequently investigated
using 13C solid-state NMR [16]. This early research demonstrated
that solid-state NMR was useful for lignin structural
characterization. The use of a proper lignin standard in the current
study clearly demonstrates that 13C CP/MAS ssNMR spectroscopy
can provide fast, accurate and quantitative determination of the
lignin content of lignocellulose biomass