Characterizing the Chemistry of Lignin Using Size-Exclusion Chromatography: An Interview with Alena Kubatova


Plants require a strong cell wall that can provide them the structural support they need to transport water and nutrients within the plant. That is why lignin, which is an organic polymer commonly found in cell walls in plants, is essential to their survival (1). Apart from providing structure, lignin helps protect the plant from disease by reinforcing the cell walls and ensuring structural integrity. Recently, lignin was discovered to possess other benefits as well, particularly in industry and manufacturing. The extraction of lignin is noted to being beneficial in the production of biofuels and paper, showcasing its potential as a renewable resource (1).

Alena Kubatova, a professor in the Department of Chemistry at the University of North Dakota, in Grand Forks, North Dakota, is exploring new approaches in characterizing lignin, to understand its structural complexities and how these methods can help improve processing in biorefineries (1). LCGC spoke to Kubatova to discuss her recent research in understanding the chemistry of lignin.

Dr. Alena Kubatova of the University of North Dakota | © Photo Credit: Alena Kubatova

Dr. Alena Kubatova of the University of North Dakota | © Photo Credit: Alena Kubatova

Would you tell us specifically why there is a resurgence of interest in characterizing the chemistry of lignin?

Lignin utilization presents a challenge that periodically resurfaces because of the goal to develop new renewable resources. This is a challenge that we, as a scientific community, have not been able to address so far.

Lignin is an abundant heteropolymer, representing approximately 30% of lignocellulosic or woody biomass. While cellulose within the lignocellulosic biomass is widely and routinely used as a feedstock to produce paper or ethanol, the remaining lignin is not effectively utilized. Lignin’s heavy aromatic content suggests a high potential for lignin utilization as a source of renewable chemicals and fuel replacing those produced by the petroleum industry. Although numerous attempts have been made to find applications for lignin, its recalcitrance limits its successful use. I think the heart of the problem with lignin utilization is a lack of understanding of its structure. This issue is further magnified as lignin's chemical structure varies in different biomass sources (for example, pine vs. switchgrass) and the processing of native lignin within plants (for example, lignin from paper industry vs. that from biorefineries).

How do you understand the significance of employing multiple methods, such as size-exclusion chromatography (SEC), thermal carbon analysis (TCA), thermal desorption-pyrolysis gas chromatography–mass spectrometry (TD-Py-GC–MS), and 31P nuclear magnetic resonance (NMR) spectroscopy, in unraveling the structural complexities of lignin?

Historically, a suite of methods has been used to characterize lignin, each offering different structural information, yet each having clear limitations. A better understanding of specific structural features of lignin and the accessibility of its certain functional groups can be provided through new analytical approaches, ultimately enabling the development of effective lignin utilization methods.

In our work, we aim to differentiate lignin feedstock from the products generated because of its treatment. Typically, the products are of a lower molecular weight (MW) than that of the original lignin. As a result, differentiation by MW seems to be the first logical step in deciphering the structural features. Therefore, size-exclusion chromatography (SEC) is the logical choice used in lignin structure elucidation. The key SEC concept is using the molecular sieve effect for separation; however, the limitation of the method may be that it assumes no chemical interactions between the chromatographic column and lignin, whereas in reality, such interactions are doomed to occur. Thus, we have used preparative SEC to fractionate lignin, the approach used previously by others. However, in our work, this method is coupled and verified with independent characterization techniques. This setup allowed us to evaluate the effectiveness of lignin fractionation.

The study highlights altered behavior in fractionated lignin (1). How might these structural revelations impact the practical application of lignin in various industries, particularly in biorefineries?

We believe the key to lignin utilization is the accessibility of specific functional groups, which will define the reactivity of native lignin or its products. Once we can account for the accessibility of the main functional groups, we may be able to determine the pertinent reaction rates and pathways to lignin's utilization.

Could you elaborate on the observed disruption in intermolecular associations of lignin after size-exclusion fractionation? How might this understanding contribute to more efficient lignin processing methods?

We characterized the SEC fractions using TCA and TD-Py-GC–MS, allowing us to separate the lignin constituents by volatility beyond the common chromatographic separation. These techniques confirmed that SEC could not fractionate lignin based solely on MW, suggesting the adsorption of small MW species on large MW compounds. Furthermore, 31P NMR spectroscopy revealed a higher level of hydroxylation in the obtained fractions than in the initial lignin. These results led us to a hypothesis that technical lignin in its initial form has a complex three-dimensional (3D) structure held together by intramolecular forces—that is, most likely, by hydrogen bonding. Some of this structure is unraveled upon fractionation, revealing hydroxyl groups that were inaccessible before fractionation.

The study notes poorly accessible hydroxyl groups in the "unraveled" lignin. How significant is this finding, and how might it influence approaches to modifying or utilizing lignin for various applications?

Hydroxyl groups provide intramolecular interactions that are among the strongest, and so they can be considered primary reactive sites. Their accessibility may be critical to any chemical process leading to lignin utilization.

Could you discuss the substantial difference in char production (the residue of plant mass heated in the absence of air) between the fractionated and non-fractionated lignin upon pyrolysis? What implications might this have in terms of energy production or other industrial uses?

At high pyrolytic temperatures, we observed significant charring for the initial lignin. By contrast, for fractionated lignin, we observed instead the production of carbon dioxide and water, which are indicators of combustion (namely, self-combustion in application to this anoxic system). These results confirm our hypothesis regarding the accessibility of hydroxyl and other oxygen-containing functional groups within the lignin structure. In the initial structure, inaccessible oxygen leads to the aforementioned charring. Yet in fractionated lignin, the oxygen (within the hydroxyl groups) can be more easily released, allowing for lignin "self-combustion." One of the current lignin applications, although not very effective, is burning lignin as an energy source; more effective combustion of unraveled lignin could potentially increase the effectiveness of this process.

Considering the findings related to altered lignin structure post-fractionation, how feasible do you think it is to implement these discoveries in real-world lignin processing and utilization in biorefineries or related industries?

We believe that we and the science community still have a long way to go before implementing our findings in real-world lignin processing. We have suggested an unraveling process and referred to a complex 3D structure of lignin held together by intramolecular forces. This, in a way, may resemble that of proteins built from amino acids. In lignomic research, structural building blocks have been investigated. Yet, unlike proteomic research, we do not yet have systematic approaches/databases to structural elucidation. Thus, we need to build towards it, which is a gradual process requiring further work.


(1) LaVallie, A.; Andrianova, A. A.; Schumaker, J.; Reagen, S.; Lu, S.; Smoliakova, I. P.; Kozliak, E. I.; Kubatova, A. Unfolding of Lignin Structure Using Size-Exclusion Fractionation. Polymers 2023, 15 (19), 3956. DOI: 10.3390/polym15193956

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