High-Resolution Analytical Separation and Structural Probes for Studies of High Molecular Mass Material From Bleached Kraft Mill Effluent, 1992 Environmental Conference Proceedings

The high molecular mass component (HMM) of BKME which is comprised largely of lignin breakdown products, is generally considered to have a complex, if not amorphous, primary chemical structure. This feature, plus the fact that HMM is a waste byproduct with no established property or function to test for, makes it difficult to detect inadvertent structural alterations during separation and isolation. Using aquatic toxicity and mode-of-action as properties of HMM, we employed mild and nondegradative procedures adapted from biomacromolecular separation technology. Following lyophilization to concentrate BKME and sequential organic solvent extraction to remove the low molecular mass hydrophobic compounds, the majority of the remaining material was found to be water-only soluble. This hydrophilic residue was dissolved in water, from which HMM is isolated by nanofiltration on hydrophilic membranes. The ultrafiltered fraction was further separated by polyacrylamide gel electrophoresis (native, SDS, and isoelectrofocusing). The resulting sharp bands are recoverable by electroelution for toxicity testing and structural studies. Structural probes for these bands included 1H NMR spectroscopy, mass spectrometry, and UV-visible spectrophotometry. From the analytical standpoint, it appears at the current time that electrophoretic separation techniques and chromophoric probes (e.g. Alcian blue) may be useful for the determination of bioactive HMM.