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

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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.

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About the authors

O. A. Trubetskoy

Institute of Basic Biological Problems of the Russian Academy of Sciences

Author for correspondence.
Email: olegi03@yahoo.com
Russian Federation, Pushchino

O. E. Trubetskaya

Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: trub@bibch.ru
Russian Federation, Pushchino

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2. Fig. 1. A multi-stage scheme of fractionation and analysis of SRO.

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3. Fig. 2. Three-dimensional fluorescence spectra of the initial drug CPO and fractions A, B and C + D obtained using EC – EPAG. The fluorescence intensity is indicated by different colors in the upper right corner of each figure.

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4. Fig. 3. RP-HPLC of SRO preparations, fractions A, B, C + D and authentic tyrosine and tryptophan amino acids with registration of fluorescence intensity at λ3 / λsp - 270 nm / 330 nm (solid line) and λ3 / λisp - 270 nm / 450 nm (broken line).

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5. Fig. 4. Fluorescence spectra of peaks 1–7, 1a, 3a, and 4a obtained during RP – HPLC of CPO preparations and fractions A, B, C + D. The spectra were extracted from the data of a multiwave fluorescence detector at an elution time corresponding to the peak of the corresponding peak.  

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