Analysis of Fluorophores of Dissolved Organic Matter in the Suwannee River by Reversed-Phase Liquid Chromatography

Abstract

Reversed-phase high performance liquid chromatography in combination with a multiwave fluorescence detector has been used to analyze the natural water of the Suwannee River for dissolved organic matter and its stable electrophoretic fractions A, B, and C + D, obtained by a combination of preparative exclusive chromatography with analytical electrophoresis in polyacrylamide gel. The fraction A had the largest molecular size, while that of the fraction C + D was the smallest. Three-dimensional fluorescent analysis revealed humic-like fluorescence both in the source preparation and in all fractions, while protein-like fluorescence was almost completely localized in fractions A and B with the largest and medium molecular size. A wide peak of humic-like fluorescence was found to split into several groups of fluorophores with different emission maximums (435, 455, 460, and 465 nm) and hydrophobic properties. The obtained results are discussed in the context of the modern theories describing the formation of humic-like fluorescence of dissolved organic matter. Identified in the fractions A and B of the largest molecular size were low-molecular free aromatic amino acids tyrosine and tryptophan, which account for >50% of the protein-like fluorescence of dissolved organic matter in Suwannee water. The obtained data contribute to the understanding of the molecular nature of protein-like and humic-like fluorescence of dissolved organic matter in natural water.