Antioxidant profiling by two-dimensional chromatography with post-column ABTS: example of lignin

Lignin as a natural antioxidant is currently gaining interest as a renewable substitute for fossil-based antioxidants. Here, two-dimensional liquid chromatography (size-exclusion and reversed-phase chromatography) coupled with a post-column radical scavenging assay (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)) is used to profile the antioxidant activity of softwood Kraft lignin. This method can assign antioxidant activity to different molecular weight fractions (monomeric, oligomeric, polymeric) within the complex lignin matrix. Four guaiacyl-type monomeric compounds (guaiacol, acetovanillone, vanillic acid, and vanillin) were identified in the lignin sample, consistent with softwood lignin composition; however, antioxidant activity varies widely among them. Guaiacol shows the highest specific radical scavenging activity, whereas vanillin's activity is lower than the detection limit. Antioxidant activity was also detected for oligomeric and polymeric domains. For polymeric lignin higher specific antioxidant activity was detected with decreasing molecular weight. This approach provides a more detailed understanding of lignin's structure-property relationship regarding its antioxidant activity than conventional bulk-type assays or solvent fractionation experiments, thereby guiding the selection of lignin fractions for antioxidant applications.