7.5.3.1 Klason, acetyl bromide, and thioglycolic acid estimations

Of these three procedures, the two most commonly used for estimating gross lignin con­tents are the Klason and AcBr methods, respectively. The first method, albeit very routinely applied, is most uninformative, since it only measures insoluble material remaining follow­ing “digestion” with 72% H2SO4 (179). While generally reliable for mature woody plant stem material, it has substantial limitations when generically applied to both herbaceous and immature woody tissues [discussed in Anterola and Lewis (77) and references therein], as well as for other tissues, such as bark. Indeed, as long ago as 1986, Leary et al. (180) carried out Klason lignin analyses on 15 samples of hardwoods, softwoods, and grasses; it was determined from these studies that various non-lignin components, such as tannins, were present in the “lignin” isolated thereby making the analyses ofsolely insoluble material suspect for various sample types.

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Figure 7.10 Sulphonation, alkaline nitrobenzene oxidation, and thioacidolysis products. (A) Sulphonated lignin-derived monomers (38-41) (147). (B) Products formed by alkaline nitrobenzene oxidation of 8-0- 4′-linked model compounds 42-44. (C) Proposed mechanism for NBO lignin cleavage. [Adapted from Schultz and co-workers (189, 190).] (D) Thioacidolysis monomeric products 54-56 from 8-0-4′ model compounds 51-53. (E) Maximum amounts (and ranges) of monomeric/dimeric products from lignins that are typically released by alkaline nitrobenzene oxidation (NBO), (71, 132) thioacidolysis (71, 132) and permanganate oxidation;the bulk of the lignin biopolymer is largely unaccounted for.

Two recently published examples ofKlason lignin contents in juvenile poplar and tobacco stems serve to further illustrate the current state of disarray in acquiring reliable basic analytical data: A report of “lignin” contents in immature (approximately 1-year old) poplar (Populus tremula x Populus alba) stems gave values as high as ~32% (181), whereas others analyzing 3-month and 2-year-old poplar had indicated that levels were ~20% (182-184). Values of ~32% are well outside the ranges expected, since it is known that, for example, mature poplar wood tissues only have lignin levels ~ 18—21% (5). If lignin contents can be overestimated by up to nearly 60%, such approaches are unlikely to identify meaningful trends in lignin deposition/composition and assembly proper, as well as on the kinetics of lignin removal. Another quite similar example also occurred in tobacco stem analyses (69), whose sections were reported to contain ~40—50% lignin rather than the 20—25% expected. Both examples reflect simply a degree of unreliability in the data obtained through Klason lignin analyses, and thus a departure from the analytical rigor expected.

Similar concerns about the unreliability of thioglycolic acid lignin determinations have also been noted and critically evaluated (77). Two examples will again suffice: thioglycolic acid levels of presumed lignin contents in stem sections of 4CL downregulated Arabidopsis stems were considered to be ~50% lower than that of wild-type levels (185). On the other hand, reanalysis of this study by Anterola and Lewis (77) established that their alkaline nitrobenzene oxidation protocols had given > 115% recoveries of lignin-derived fragments, rather than the ~25% or so expected, and suggest unreliability in one or both procedures. These results again reflect significant departure from the data expected. A similar level of unreliability was also noted for COMT downregulated alfalfa analyses: lignin contents were considered to be reduced by ~50% (186), although many other studies (77) have demonstrated that there were no significant levels of reduction in lignin amounts following COMT downregulation.

The AcBr method “solubilizes” lignins, as well as other non-lignin phenolics, as bromi — nated derivatives; it has also been applied to numerous lignin determinations, using a generic extinction coefficient of e280 = 20.09Lg-1cm-1 (179,187). Again, while very routinely used, this method does not take into account the differences in extinction coefficients due to vari­ations in lignin monomeric H, G, and S compositions. In our more recent investigations using H-, G-, and S-enriched lignin isolates, the best current estimates of the actual extinction coefficients (, 280 nm) were established to be considerably different, i. e., (H) 15.3, (G) 18.6, and (S) 14.6 L g-1cm-1 for p-coumaryl (1), coniferyl (3), and sinapyl (5) alcohol-enriched lignins (71,188). These differences thus again underscore the need for both circumspection and scientific rigor in the study of lignins, i. e., as routinely expected for all other areas of natural product chemistry.