High amounts of free aromatic amino acids in the protein-like fluorescence of water-dissolved organic matter

International audienceFluorescence spectroscopy is widely used to study water pollution. The fluorescence of water natural organic matter can be classified into two groups: the protein-like fluorescence originating from aromatic amino acids and the humic fluorescence originating from humic substances. Actually, the precise molecular origin of the protein-like fluorescence is unknown because this fluorescence may be caused by either free amino acids, peptides or proteins. Therefore, we studied the molecular origin of the protein-like fluorescence of Suwannee River natural organic matter and fractions A, B and C + D obtained by size exclusion chromatography/polyacrylamide gel electrophoresis. Fractions were analyzed by reversed-phase high-performance liquid chromatography. The electrophoretic mobilities of fractions varied in the order C + D > B > A and the molecular size in the opposite order. Our results show that the protein-like fluorescence is almost exclusively located in high molecular size fraction A and medium molecular size fraction B. Retention times and fluorescence emission spectra of authentic free aromatic amino acids tyrosine and tryptophan were identical with the retention times and emission spectra of several chromatographic peaks of fractions A and B. More than 50 % of the protein-like fluorescence is due to free aromatic amino acids incorporated in water natural organic matter

Evaluation of Suwannee River NOM electrophoretic fractions by RP-HPLC with online absorbance and fluorescence detection.

International audienceReversed-phase high-performance liquid chromatography (RP-HPLC) with online absorbance and fluorescence detections was used for the evaluation of Suwannee River natural organic matter (SRNOM) and its fractions A, B, and C+D, obtained by conventional size exclusion chromatography–polyacrylamide gel electrophoresis (SEC-PAGE) setup, for which the electrophoretic mobility (EM) and the absorptivity varied in the order C+D > B > A, and the molecular size (MS) in the opposite order. Analysis of SRNOM and its fractions in part of their relative irreversible adsorption on C18-column and relative distribution of eluted from the column matter on hydrophobic and hydrophilic peaks showed that hydrophobicity of fractions decreased in order: A > B > C+D. The online fluorescence detection showed that SRNOM and its fractions contained at least three groups of humic substances (HS)-like fluorophores with emission maxima at 435, 455–465, and 455/420 nm and two protein-like fluorophores with emission maxima at around 300 and 340 nm. The HS-like fluorophore with emission maximum at 435 nm was located in the hydrophilic peak in all the samples. Those with maxima at 455–465 nm were detected in hydrophobic peaks of fractions A and B. SEC-PAGE setup followed by RP-HPLC allowed us to develop new approach of SRNOM separation on less heterogeneous compounds mixture for their further study and structural identification

Determination of hydrophobicity and optical properties of soil humic acids isolated by different methods

International audienceHAs were extracted from the same chernozem soil sample by two commonly used methods: (i) extraction by 0.1 M NaOH followed by pre-decalcification using 0.1 M HCl -- the method, recommended by the International Humic Substances Society, and the HAs sample was named IHSS-HAs; and (ii) Kononova's method, where several treatments of soil by 0.1 M Na4P2O7 + 0.1 M NaOH were employed and the HAs were marked K1-HAs, K2-HAs and K3-HAs (corresponding to the first, second and third isolation cycle, respectively). Reversed-phase high performance liquid chromatography (RP HPLC) with stepwise and linear gradients of methanol was used to reveal hydrophobicity differences in isolated HAs. Independent on the method used, after the first extraction IHSS-HAs and K1-HAs had a similar hydrophobicity, and varied in the order K3-HAs > K2-HAs > K1-HAs in Kononova's method. The absorption spectra of extracted HAs were featureless, but the negative correlation of specific absorption at 280 nm and E4/E6 ratio with hydrophobicity was found. The data point out that the hydrophobicity of HAs may be greatly influenced by their isolation procedure. Extraction protocol may induce changes in the transport of organic pollutants and affected drawn conclusions

Excitation-Dependent Fluorescence Quantum Yield for Freshwater Chromophoric Dissolved Organic Matter from Northern Russian Lakes

Advanced fluorescence analysis within the wide range of excitation wavelengths from 230 to 510 nm accompanied with chromatography was used to study natural chromophoric dissolved organic matter (CDOM) from three freshwater Karelian lakes. The influence of excitation wavelength (λex) on fluorescence quantum yield and emission maximum position was determined. The CDOM fluorescence quantum yield has reached a minimum at λex∼270–280 nm and a maximum at λex∼340–360 nm. It was monotonously decreasing after 370 nm towards longer excitation wavelengths. Analytical reversed-phase high-performance liquid chromatography with multiwavelength fluorescence detector characterized distribution of fluorophores between hydrophilic/hydrophobic CDOM parts. This technique revealed “hidden” protein-like fluorophores for some CDOM fractions, in spite of the absence of protein-like fluorescence in the initial CDOM samples. The humic-like fluorescence was documented for all hydrophilic and hydrophobic CDOM chromatographic peaks, and its intensity was decreasing along with peaks’ hydrophobicity. On contrary, the protein-like fluorescence was found only in the hydrophobic peaks, and its intensity was increasing along with peaks’ hydrophobicity. This work provides new data on the CDOM optical properties consistent with the formation of supramolecular assemblies controlled by association of low-molecular size components. In addition, these data are very useful for understanding the CDOM function in the environment

RP-HPLC and spectroscopic characterization of Suwannee River water NOM after concentrated urea treatment and dialysis

International audienceSuwannee River natural organic matter (SRNOM) was treated by 7 M urea and then purified by dialysis on 10 kDa membrane. The untreated SRNOM and treated (USRNOM) samples were examined using UV–visible and fluorescence spectroscopies and reversed-phase high-performance liquid chromatography (RP-HPLC) with online absorbance and fluorescence detection. USRNOM was 1.5-fold more absorbing at 280 nm than SRNOM and four fold less fluorescent than SRNOM upon excitation at 270 nm. RP-HPLC analyses of the two samples revealed that USRNOM was somewhat more hydrophobic than SRNOM and both samples contained at least two groups of HS-like fluorophores with different hydrophobicity and protein-like fluorophore(s). Data indicate that protein-like fluorophores were not lost during dialysis. They showed hydrophobic properties and seemed highly fluorescent. HS-like and protein-like fluorophores from water NOM could be successfully separated by RP-HPLC. This raises the prospect of their further research and identification and could be significant for future NOM chemical structure characterization