Analysis of fluorophores of organic substances dissolved in Suvani river water using reversed-phase liquid chromatography

Abstract

Reversed-phase high-efficiency liquid chromatography was used in combination with detection by multiwave fluorescence for analysis of organic substances dissolved in natural water of the Suwannee River. Also analyzed were the stable electrophoretic fractions А, В, and C+D, obtained by a combination of preparative size-exclusion chromatography and analytical electrophoresis in a polyacrylamide gel. Fraction А has the largest molecular size, and fraction C+D, the smallest. Using 3D fluorescent analysis, humic-like fluorescence was detected both in the original sample and in all fractions; protein-like fluorescence is almost fully localized in fractions А and В of the largest and middle molecular sizes. The wide peak of humic-like fluorescence is split into several groups of fluorophores with different emissions maxima (435, 455, 460, and 465 nm) and degrees of hydrophobicity. The obtained results were analyzed in relation to contemporary theories of formation of humic-like fluorescence of dissolved organic substances. The low-molecular free aromatic amino acids tyrosine and tryptophan were identified in fractions А and В of the highest molecular size and constitute >50% of the protein-like fluorescence of the organic substances dissolved in the Suwannee River water. The data obtained ensure better understanding of the molecular nature of protein-like and humic-like fluorescence of organic substance dissolved in natural water.