RESUMO
The study of dissolved organic matter (DOM) presents a significant challenge for environmental analyses and the monitoring of wastewater treatment plants (WWTPs). This is particularly true for the tracking of recalcitrant to biodegradation dissolved organic matter (rDOM) compounds, which is generated during the thermal pretreatment of sludge. This study aims to develop analytical and chemometric methods to differentiate melanoidins from humic acids (HAs), two components of rDOM that require monitoring at various stages of wastewater treatment processes due to their distinct biological effects. The developed method implements the separation of macromolecules through ultra-high-performance liquid chromatography size-exclusion chromatography (U-HPLC SEC) followed by online UV and fluorescence detection. UV detection was performed at 210, 254, and 280 nm, and fluorescence detection at six excitation/emission pairs: 230/355 nm, 270/355 nm, 240/440 nm, 270/500 nm, 330/425 nm, and 390/500 nm. Chromatograms obtained for each sample from these nine detection modes were integrated and separated into four molecular fractions: >40 kDa, 20-40 kDa, 10-20 kDa, and <10 kDa. To enhance analytical resolution and normalize the data, ratios were calculated from the areas of chromatographic peaks obtained for each detection mode. The results demonstrate the utility of these ratios in discriminating samples composed of HAs, melanoidins, and their mixtures, through principal component analysis (PCA). Low molecular weight fractions were found to be specific to melanoidins, while high molecular weight fractions were characteristic of HAs. For the detection modes specific to melanoidins, UV absorbance at 210, 254, and 280 nm were predominantly present in the numerators, with tryptophan-like fluorescence emissions in the denominators. Conversely, fluorescence emissions largely represented both numerators and denominators for HAs. This online method also enables the discrimination of pseudo-melanoidins, compounds revealing a nitrogen deficiency in their chemical structures.
RESUMO
The study of organic matter in wastewater is a major regulatory and environmental issue and requires new developments to identify non-biodegradable refractory compounds, produced mainly by thermal treatments. Recent advances linking physicochemical properties to spectroscopic analyzes (UV, Fluorescence, IR) have shown that the refractory property is favored by several physicochemical parameters: weight, hydrophobicity, aromaticity and chemical functions. Currently, the most effective developments for the quantification of refractory compounds are obtained with hyphenated methods, based on steric separation of the macromolecular species by steric exclusion chromatography (SEC)/PDA/Fluorescence systems. Hyphenated techniques using High Resolution Mass Spectrometry (HRMS), ultra-high-resolution mass spectrometry with Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and NMR have been developed to analyze macromolecules in wastewater with minor sample preparation procedures. A particular class has been identified, the melanoidins, generated by Maillard reactions between sugars, amino acids, peptides and proteins present in wastewater and sludge, but low molecular weight compounds formed as intermediates, such as ketones, aldehydes, pyrazines, pyridines or furans, are also recalcitrant and are complex to identify in the complex matrices. The lack of available standards for the study of these compounds requires the use of specific techniques and data processing. Advances in chemometrics are obtained in the development of molecular or physicochemical indices resulting from the data generated by the analytical detectors, such as aromaticity calculated by SUVA254 and determined by UV, fluorescence, molar mass, H/C ratio or structural studies (measuring the amount of unsaturated carbon) given by hyphenated techniques with SEC. It is clear that nitrogen compounds are widely involved in refractoriness. New trends in nitrogen containing compounds characterization follow two axes: through SEC/PDA/Fluorescence and HRMS/NMR techniques with or without separation. Other techniques widely used in food or marine science are also being imported to this study, as it can be seen in the use of "omics" methods, high-performance thin layer chromatography (HPTLC) and chromatography at the critical condition, rounding out the important developments around SEC. While improving the performance of stationary phases is one of the challenges, it results in a fundamental understanding of the retention mechanisms that today provide us with more information on the structures identified. The main objective of this review is to present the spectroscopic and physicochemical techniques used to qualify and characterize refractoriness with a specific focus on chemometric approaches.